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Where do the phosphate units come from when EGF units dimerize?

Where do the phosphate units come from when EGF units dimerize?


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Upon EGF binding, EGFR units dimerize and cross-phosphorylate. The phosphate groups are transfered to intracellular c-terminal tyrosine rich regions. Where do the phosphate units come from in this cross-reaction? Is it from bound GTP units?


In protein phosphorylation, the phosphate group transferred from the kinase to the substrate comes almost universally from ATP. However, there is evidence of some protein kinases using GTP as their phosphate source, although they are few and far between.


Guanine nucleotide exchange factor

Guanine nucleotide exchange factors (GEFs) are proteins or protein domains that activate monomeric GTPases by stimulating the release of guanosine diphosphate (GDP) to allow binding of guanosine triphosphate (GTP). [1] A variety of unrelated structural domains have been shown to exhibit guanine nucleotide exchange activity. Some GEFs can activate multiple GTPases while others are specific to a single GTPase.


Contents

Initially, transcription of alternative splice variants derived from the INSR gene are translated to form one of two monomeric isomers IR-A in which exon 11 is excluded, and IR-B in which exon 11 is included. Inclusion of exon 11 results in the addition of 12 amino acids upstream of the intrinsic furin proteolytic cleavage site.

Upon receptor dimerisation, after proteolytic cleavage into the α- and β-chains, the additional 12 amino acids remain present at the C-terminus of the α-chain (designated αCT) where they are predicted to influence receptor–ligand interaction. [9]

Each isometric monomer is structurally organized into 8 distinct domains consists of a leucine-rich repeat domain (L1, residues 1-157), a cysteine-rich region (CR, residues 158-310), an additional leucine rich repeat domain (L2, residues 311-470), three fibronectin type III domains FnIII-1 (residues 471-595), FnIII-2 (residues 596-808) and FnIII-3 (residues 809-906). Additionally, an insert domain (ID, residues 638-756) resides within FnIII-2, containing the α/β furin cleavage site, from which proteolysis results in both IDα and IDβ domains. Within the β-chain, downstream of the FnIII-3 domain lies a transmembrane helix (TH) and intracellular juxtamembrane (JM) region, just upstream of the intracellular tyrosine kinase (TK) catalytic domain, responsible for subsequent intracellular signaling pathways. [10]

Upon cleavage of the monomer to its respective α- and β-chains, receptor hetero or homo-dimerisation is maintained covalently between chains by a single disulphide link and between monomers in the dimer by two disulphide links extending from each α-chain. The overall 3D ectodomain structure, possessing four ligand binding sites, resembles an inverted 'V', with the each monomer rotated approximately 2-fold about an axis running parallel to the inverted 'V' and L2 and FnIII-1 domains from each monomer forming the inverted 'V's apex. [10] [11]

The insulin receptor's endogenous ligands include insulin, IGF-I and IGF-II. Using a cryo-EM, structural insight into conformational changes upon insulin binding was provided. Binding of ligand to the α-chains of the IR dimeric ectodomain shifts it from an inverted V-shape to a T-shaped conformation, and this change is propagated structurally to the transmembrane domains, which get closer, eventually leading to autophosphorylation of various tyrosine residues within the intracellular TK domain of the β-chain. [12] These changes facilitate the recruitment of specific adapter proteins such as the insulin receptor substrate proteins (IRS) in addition to SH2-B (Src Homology 2 - B ), APS and protein phosphatases, such as PTP1B, eventually promoting downstream processes involving blood glucose homeostasis. [14]

Strictly speaking the relationship between IR and ligand shows complex allosteric properties. This was indicated with the use of a Scatchard plots which identified that the measurement of the ratio of IR bound ligand to unbound ligand does not follow a linear relationship with respect to changes in the concentration of IR bound ligand, suggesting that the IR and its respective ligand share a relationship of cooperative binding. [15] Furthermore, the observation that the rate of IR-ligand dissociation is accelerated upon addition of unbound ligand implies that the nature of this cooperation is negative said differently, that the initial binding of ligand to the IR inhibits further binding to its second active site - exhibition of allosteric inhibition. [15]

These models state that each IR monomer possesses 2 insulin binding sites site 1, which binds to the 'classical' binding surface of insulin: consisting of L1 plus αCT domains and site 2, consisting of loops at the junction of FnIII-1 and FnIII-2 predicted to bind to the 'novel' hexamer face binding site of insulin. [5] As each monomer contributing to the IR ectodomain exhibits 3D 'mirrored' complementarity, N-terminal site 1 of one monomer ultimately faces C-terminal site 2 of the second monomer, where this is also true for each monomers mirrored complement (the opposite side of the ectodomain structure). Current literature distinguishes the complement binding sites by designating the second monomer's site 1 and site 2 nomenclature as either site 3 and site 4 or as site 1' and site 2' respectively. [5] [14] As such, these models state that each IR may bind to an insulin molecule (which has two binding surfaces) via 4 locations, being site 1, 2, (3/1') or (4/2'). As each site 1 proximally faces site 2, upon insulin binding to a specific site, 'crosslinking' via ligand between monomers is predicted to occur (i.e. as [monomer 1 Site 1 - Insulin - monomer 2 Site (4/2')] or as [monomer 1 Site 2 - Insulin - monomer 2 site (3/1')]). In accordance with current mathematical modelling of IR-insulin kinetics, there are two important consequences to the events of insulin crosslinking 1. that by the aforementioned observation of negative cooperation between IR and its ligand that subsequent binding of ligand to the IR is reduced and 2. that the physical action of crosslinking brings the ectodomain into such a conformation that is required for intracellular tyrosine phosphorylation events to ensue (i.e. these events serve as the requirements for receptor activation and eventual maintenance of blood glucose homeostasis). [14]

Applying cryo-EM and molecular dynamics simulations of receptor reconstituted in nanodiscs, the structure of the entire dimeric insulin receptor ectodomain with four insulin molecules bound was visualized, therefore confirming and directly showing biochemically predicted 4 binding locations. [13]

Agonists Edit

The Insulin Receptor is a type of tyrosine kinase receptor, in which the binding of an agonistic ligand triggers autophosphorylation of the tyrosine residues, with each subunit phosphorylating its partner. The addition of the phosphate groups generates a binding site for the insulin receptor substrate (IRS-1), which is subsequently activated via phosphorylation. The activated IRS-1 initiates the signal transduction pathway and binds to phosphoinositide 3-kinase (PI3K), in turn causing its activation. This then catalyses the conversion of Phosphatidylinositol 4,5-bisphosphate into Phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 acts as a secondary messenger and induces the activation of phosphatidylinositol dependent protein kinase, which then activates several other kinases – most notably protein kinase B, (PKB, also known as Akt). PKB triggers the translocation of glucose transporter (GLUT4) containing vesicles to the cell membrane, via the activation of SNARE proteins, to facilitate the diffusion of glucose into the cell. PKB also phosphorylates and inhibits glycogen synthase kinase, which is an enzyme that inhibits glycogen synthase. Therefore, PKB acts to start the process of glycogenesis, which ultimately reduces blood-glucose concentration. [16]

Effect of insulin on glucose uptake and metabolism. Insulin binds to its receptor (1), which, in turn, starts many protein activation cascades (2). These include: translocation of Glut-4 transporter to the plasma membrane and influx of glucose (3), glycogen synthesis (4), glycolysis (5), and fatty acid synthesis (6).

Signal transduction of Insulin: At the end of the transduction process, the activated protein binds to the PIP2 proteins embedded in the membrane.

Regulation of gene expression Edit

The activated IRS-1 acts as a secondary messenger within the cell to stimulate the transcription of insulin-regulated genes. First, the protein Grb2 binds the P-Tyr residue of IRS-1 in its SH2 domain. Grb2 is then able to bind SOS, which in turn catalyzes the replacement of bound GDP with GTP on Ras, a G protein. This protein then begins a phosphorylation cascade, culminating in the activation of mitogen-activated protein kinase (MAPK), which enters the nucleus and phosphorylates various nuclear transcription factors (such as Elk1).

Stimulation of glycogen synthesis Edit

Glycogen synthesis is also stimulated by the insulin receptor via IRS-1. In this case, it is the SH2 domain of PI-3 kinase (PI-3K) that binds the P-Tyr of IRS-1. Now activated, PI-3K can convert the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3). This indirectly activates a protein kinase, PKB (Akt), via phosphorylation. PKB then phosphorylates several target proteins, including glycogen synthase kinase 3 (GSK-3). GSK-3 is responsible for phosphorylating (and thus deactivating) glycogen synthase. When GSK-3 is phosphorylated, it is deactivated, and prevented from deactivating glycogen synthase. In this roundabout manner, insulin increases glycogen synthesis.

Degradation of insulin Edit

Once an insulin molecule has docked onto the receptor and effected its action, it may be released back into the extracellular environment or it may be degraded by the cell. Degradation normally involves endocytosis of the insulin-receptor complex followed by the action of insulin degrading enzyme. Most insulin molecules are degraded by liver cells. It has been estimated that a typical insulin molecule is finally degraded about 71 minutes after its initial release into circulation. [17]

Immune system Edit

Besides the metabolic function, insulin receptors are also expressed on immune cells, such as macrophages, B cells, and T cells. On T cells, the expression of insulin receptors is undetectable during the resting state but up-regulated upon T-cell receptor (TCR) activation. Indeed, insulin has been shown when supplied exogenously to promote in vitro T cell proliferation in animal models. Insulin receptor signalling is important for maximizing the potential effect of T cells during acute infection and inflammation. [18] [19]

The main activity of activation of the insulin receptor is inducing glucose uptake. For this reason "insulin insensitivity", or a decrease in insulin receptor signaling, leads to diabetes mellitus type 2 – the cells are unable to take up glucose, and the result is hyperglycemia (an increase in circulating glucose), and all the sequelae that result from diabetes.

A few patients with homozygous mutations in the INSR gene have been described, which causes Donohue syndrome or Leprechaunism. This autosomal recessive disorder results in a totally non-functional insulin receptor. These patients have low-set, often protuberant, ears, flared nostrils, thickened lips, and severe growth retardation. In most cases, the outlook for these patients is extremely poor, with death occurring within the first year of life. Other mutations of the same gene cause the less severe Rabson-Mendenhall syndrome, in which patients have characteristically abnormal teeth, hypertrophic gingiva (gums), and enlargement of the pineal gland. Both diseases present with fluctuations of the glucose level: After a meal the glucose is initially very high, and then falls rapidly to abnormally low levels. [20] Other genetic mutations to the insulin receptor gene can cause Severe Insulin Resistance. [21]


Contents

HER2 is so named because it has a similar structure to human epidermal growth factor receptor, or HER1. Neu is so named because it was derived from a rodent glioblastoma cell line, a type of neural tumor. ErbB-2 was named for its similarity to ErbB (avian erythroblastosis oncogene B), the oncogene later found to code for EGFR. Molecular cloning of the gene showed that HER2, Neu, and ErbB-2 are all encoded by the same orthologs. [9]

ERBB2, a known proto-oncogene, is located at the long arm of human chromosome 17 (17q12).

The ErbB family consists of four plasma membrane-bound receptor tyrosine kinases. One of which is erbB-2, and the other members being epidermal growth factor receptor, erbB-3 (neuregulin-binding lacks kinase domain), and erbB-4. All four contain an extracellular ligand binding domain, a transmembrane domain, and an intracellular domain that can interact with a multitude of signaling molecules and exhibit both ligand-dependent and ligand-independent activity. Notably, no ligands for HER2 have yet been identified. [10] [11] HER2 can heterodimerise with any of the other three receptors and is considered to be the preferred dimerisation partner of the other ErbB receptors. [12]

Dimerisation results in the autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptors and initiates a variety of signaling pathways.

Signal transduction Edit

Signaling pathways activated by HER2 include: [13]

In summary, signaling through the ErbB family of receptors promotes cell proliferation and opposes apoptosis, and therefore must be tightly regulated to prevent uncontrolled cell growth from occurring.

Cancer Edit

Amplification, also known as the over-expression of the ERBB2 gene, occurs in approximately 15-30% of breast cancers. [8] [14] It is strongly associated with increased disease recurrence and a poor prognosis however, drug agents targeting HER2 in breast cancer have significantly positively altered the otherwise poor-prognosis natural history of HER2-positive breast cancer. [15] Over-expression is also known to occur in ovarian, [16] stomach, adenocarcinoma of the lung [17] and aggressive forms of uterine cancer, such as uterine serous endometrial carcinoma, [18] [19] e.g. HER2 is over-expressed in approximately 7-34% of patients with gastric cancer [20] [21] and in 30% of salivary duct carcinomas. [22]

HER2 is colocalised and most of the time, coamplified with the gene GRB7, which is a proto-oncogene associated with breast, testicular germ cell, gastric, and esophageal tumours.

HER2 proteins have been shown to form clusters in cell membranes that may play a role in tumorigenesis. [23] [24]

Evidence has also implicated HER2 signaling in resistance to the EGFR-targeted cancer drug cetuximab. [25]

Mutations Edit

Furthermore, diverse structural alterations have been identified that cause ligand-independent firing of this receptor, doing so in the absence of receptor over-expression. HER2 is found in a variety of tumours and some of these tumours carry point mutations in the sequence specifying the transmembrane domain of HER2. Substitution of a valine for a glutamic acid in the transmembrane domain can result in the constitutive dimerisation of this protein in the absence of a ligand. [26]

HER2 mutations have been found in non-small-cell lung cancers (NSCLC) and can direct treatment. [27]

HER2 is the target of the monoclonal antibody trastuzumab (marketed as Herceptin). Trastuzumab is effective only in cancers where HER2 is over-expressed. One year of trastuzumab therapy is recommended for all patients with HER2-positive breast cancer who are also receiving chemotherapy. [28] Twelve months of trastuzumab therapy is optimal. Randomized trials have demonstrated no additional benefit beyond 12 months, whereas 6 months has been shown to be inferior to 12. Trastuzumab is administered intravenously weekly or every 3 weeks. [29]

An important downstream effect of trastuzumab binding to HER2 is an increase in p27, a protein that halts cell proliferation. [30] Another monoclonal antibody, Pertuzumab, which inhibits dimerisation of HER2 and HER3 receptors, was approved by the FDA for use in combination with trastuzumab in June 2012.

As of November 2015, there are a number of ongoing and recently completed clinical trials of novel targeted agents for HER2+ metastatic breast cancer, e.g. margetuximab. [31]

Additionally, NeuVax (Galena Biopharma) is a peptide-based immunotherapy that directs "killer" T cells to target and destroy cancer cells that express HER2. It has entered phase 3 clinical trials.

It has been found that patients with ER+ (Estrogen receptor positive)/HER2+ compared with ER-/HER2+ breast cancers may actually benefit more from drugs that inhibit the PI3K/AKT molecular pathway. [32]

Over-expression of HER2 can also be suppressed by the amplification of other genes. Research is currently being conducted to discover which genes may have this desired effect.

The expression of HER2 is regulated by signaling through estrogen receptors. Normally, estradiol and tamoxifen acting through the estrogen receptor down-regulate the expression of HER2. However, when the ratio of the coactivator AIB-3 exceeds that of the corepressor PAX2, the expression of HER2 is upregulated in the presence of tamoxifen, leading to tamoxifen-resistant breast cancer. [33] [34]

Cancer biopsy Edit

HER2 testing is performed in breast cancer patients to assess prognosis and to determine suitability for trastuzumab therapy. It is important that trastuzumab is restricted to HER2-positive individuals as it is expensive and has been associated with cardiac toxicity. [35] For HER2-positive tumours, the risks of trastuzumab clearly outweigh the benefits.

Tests are usually performed on breast biopsy samples obtained by either fine-needle aspiration, core needle biopsy, vacuum-assisted breast biopsy, or surgical excision. Immunohistochemistry is used to measure the amount of HER2 protein present in the sample. Examples of this assay include HercepTest, Dako, Glostrup, and Denmark. The sample is given a score based on the cell membrane staining pattern.

  • Complete membrane staining that is either nonuniform or weak in intensity, but has circumferential distribution in at least 10% of cells. [37][38]

Specimens with equivocal IHC results should then be validated using fluorescence in situ hybridisation (FISH). FISH can be used to measure the number of copies of the gene which are present and is thought to be more reliable than IHC. [39]

Serum Edit

The extracellular domain of HER2 can be shed from the surface of tumour cells and enter the circulation. Measurement of serum HER2 by enzyme-linked immunosorbent assay (ELISA) offers a far less invasive method of determining HER2 status than a biopsy and consequently has been extensively investigated. Results so far have suggested that changes in serum HER2 concentrations may be useful in predicting response to trastuzumab therapy. [40] However, its ability to determine eligibility for trastuzumab therapy is less clear. [41]


Contents

The first RTKs to be discovered were EGF and NGF in the 1960s, but the classification of receptor tyrosine kinases was not developed until the 1970s. [5]

Approximately 20 different RTK classes have been identified. [6]

  1. RTK class I (EGF receptor family) (ErbB family)
  2. RTK class II (Insulin receptor family) (PDGF receptor family)
  3. RTK class IV (VEGF receptors family)
  4. RTK class V (FGF receptor family)
  5. RTK class VI (CCK receptor family)
  6. RTK class VII (NGF receptor family)
  7. RTK class VIII (HGF receptor family)
  8. RTK class IX (Eph receptor family)
  9. RTK class X (AXL receptor family)
  10. RTK class XI (TIE receptor family)
  11. RTK class XII (RYK receptor family)
  12. RTK class XIII (DDR receptor family)
  13. RTK class XIV (RET receptor family)
  14. RTK class XV (ROS receptor family)
  15. RTK class XVI (LTK receptor family)
  16. RTK class XVII (ROR receptor family)
  17. RTK class XVIII (MuSK receptor family)
  18. RTK class XIX (LMR receptor)
  19. RTK class XX (Undetermined)

Most RTKs are single subunit receptors but some exist as multimeric complexes, e.g., the insulin receptor that forms disulfide linked dimers in the presence of hormone (insulin) moreover, ligand binding to the extracellular domain induces formation of receptor dimers. [7] Each monomer has a single hydrophobic transmembrane-spanning domain composed of 25 to 38 amino acids, an extracellular N terminal region, and an intracellular C terminal region. [8] The extracellular N terminal region exhibits a variety of conserved elements including immunoglobulin (Ig)-like or epidermal growth factor (EGF)-like domains, fibronectin type III repeats, or cysteine-rich regions that are characteristic for each subfamily of RTKs these domains contain primarily a ligand-binding site, which binds extracellular ligands, e.g., a particular growth factor or hormone. [2] The intracellular C terminal region displays the highest level of conservation and comprises catalytic domains responsible for the kinase activity of these receptors, which catalyses receptor autophosphorylation and tyrosine phosphorylation of RTK substrates. [2]

In biochemistry, a kinase is a type of enzyme that transfers phosphate groups (see below) from high-energy donor molecules, such as ATP (see below) to specific target molecules (substrates) the process is termed phosphorylation. The opposite, an enzyme that removes phosphate groups from targets, is known as a phosphatase. Kinase enzymes that specifically phosphorylate tyrosine amino acids are termed tyrosine kinases.

When a growth factor binds to the extracellular domain of a RTK, its dimerization is triggered with other adjacent RTKs. Dimerization leads to a rapid activation of the protein's cytoplasmic kinase domains, the first substrate for these domains being the receptor itself. The activated receptor as a result then becomes autophosphorylated on multiple specific intracellular tyrosine residues.

Through diverse means, extracellular ligand binding will typically cause or stabilize receptor dimerization. This allows a tyrosine in the cytoplasmic portion of each receptor monomer to be trans-phosphorylated by its partner receptor, propagating a signal through the plasma membrane. [9] The phosphorylation of specific tyrosine residues within the activated receptor creates binding sites for Src homology 2 (SH2) domain- and phosphotyrosine binding (PTB) domain-containing proteins. [10] [11] Specific proteins containing these domains include Src and phospholipase Cγ. Phosphorylation and activation of these two proteins on receptor binding lead to the initiation of signal transduction pathways. Other proteins that interact with the activated receptor act as adaptor proteins and have no intrinsic enzymatic activity of their own. These adaptor proteins link RTK activation to downstream signal transduction pathways, such as the MAP kinase signalling cascade. [2] An example of a vital signal transduction pathway involves the tyrosine kinase receptor, c-met, which is required for the survival and proliferation of migrating myoblasts during myogenesis. A lack of c-met disrupts secondary myogenesis and—as in LBX1—prevents the formation of limb musculature. This local action of FGFs (Fibroblast Growth Factors) with their RTK receptors is classified as paracrine signalling. As RTK receptors phosphorylate multiple tyrosine residues, they can activate multiple signal transduction pathways.

Epidermal growth factor receptor family Edit

The ErbB protein family or epidermal growth factor receptor (EGFR) family is a family of four structurally related receptor tyrosine kinases. Insufficient ErbB signaling in humans is associated with the development of neurodegenerative diseases, such as multiple sclerosis and Alzheimer's Disease. [12] In mice, loss of signaling by any member of the ErbB family results in embryonic lethality with defects in organs including the lungs, skin, heart, and brain. Excessive ErbB signaling is associated with the development of a wide variety of types of solid tumor. ErbB-1 and ErbB-2 are found in many human cancers and their excessive signaling may be critical factors in the development and malignancy of these tumors. [13]

Fibroblast growth factor receptor (FGFR) family Edit

Fibroblast growth factors comprise the largest family of growth factor ligands at 23 members. [14] The natural alternate splicing of four fibroblast growth factor receptor (FGFR) genes results in the production of over 48 different isoforms of FGFR. [15] These isoforms vary in their ligand binding properties and kinase domains however, all share a common extracellular region composed of three immunoglobulin (Ig)-like domains (D1-D3), and thus belong to the immunoglobulin superfamily. [16] Interactions with FGFs occur via FGFR domains D2 and D3. Each receptor can be activated by several FGFs. In many cases, the FGFs themselves can also activate more than one receptor. This is not the case with FGF-7, however, which can activate only FGFR2b. [15] A gene for a fifth FGFR protein, FGFR5, has also been identified. In contrast to FGFRs 1-4, it lacks a cytoplasmic tyrosine kinase domain, and one isoform, FGFR5γ, only contains the extracellular domains D1 and D2. [17]

Vascular endothelial growth factor receptor (VEGFR) family Edit

Vascular endothelial growth factor (VEGF) is one of the main inducers of endothelial cell proliferation and permeability of blood vessels. Two RTKs bind to VEGF at the cell surface, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1). [18]

The VEGF receptors have an extracellular portion consisting of seven Ig-like domains so, like FGFRs, belong to the immunoglobulin superfamily. They also possess a single transmembrane spanning region and an intracellular portion containing a split tyrosine-kinase domain. VEGF-A binds to VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1). VEGFR-2 appears to mediate almost all of the known cellular responses to VEGF. The function of VEGFR-1 is less well defined, although it is thought to modulate VEGFR-2 signaling. Another function of VEGFR-1 may be to act as a dummy/decoy receptor, sequestering VEGF from VEGFR-2 binding (this appears to be particularly important during vasculogenesis in the embryo). A third receptor has been discovered (VEGFR-3) however, VEGF-A is not a ligand for this receptor. VEGFR-3 mediates lymphangiogenesis in response to VEGF-C and VEGF-D.

RET receptor family Edit

The natural alternate splicing of the RET gene results in the production of 3 different isoforms of the protein RET. RET51, RET43, and RET9 contain 51, 43, and 9 amino acids in their C-terminal tail, respectively. [19] The biological roles of isoforms RET51 and RET9 are the most well studied in-vivo, as these are the most common isoforms in which RET occurs.

RET is the receptor for members of the glial cell line-derived neurotrophic factor (GDNF) family of extracellular signalling molecules or ligands (GFLs). [20]

In order to activate RET, first GFLs must form a complex with a glycosylphosphatidylinositol (GPI)-anchored co-receptor. The co-receptors themselves are classified as members of the GDNF receptor-α (GFRα) protein family. Different members of the GFRα family (GFRα1-GFRα4) exhibit a specific binding activity for a specific GFLs. [21] Upon GFL-GFRα complex formation, the complex then brings together two molecules of RET, triggering trans-autophosphorylation of specific tyrosine residues within the tyrosine kinase domain of each RET molecule. Phosphorylation of these tyrosines then initiates intracellular signal transduction processes. [22]

Eph receptor family Edit

Ephrin and Eph receptors are the largest subfamily of RTKs.

Discoidin domain receptor (DDR) family Edit

The DDRs are unique RTKs in that they bind to collagens rather than soluble growth factors. [23]

The receptor tyrosine kinase (RTK) pathway is carefully regulated by a variety of positive and negative feedback loops. [24] Because RTKs coordinate a wide variety of cellular functions such as cell proliferation and differentiation, they must be regulated to prevent severe abnormalities in cellular functioning such as cancer and fibrosis. [25]

Protein tyrosine phosphatases Edit

Protein Tyrosine Phosphatase (PTPs) are a group of enzymes that possess a catalytic domain with phosphotyrosine-specific phosphohydrolase activity. PTPs are capable of modifying the activity of receptor tyrosine kinases in both a positive and negative manner. [26] PTPs can dephosphorylate the activated phosphorylated tyrosine residues on the RTKs [27] which virtually leads to termination of the signal. Studies involving PTP1B, a widely known PTP involved in the regulation of the cell cycle and cytokine receptor signaling, has shown to dephosphorylate the epidermal growth factor receptor [28] and the insulin receptor. [29] Some PTPs, on the other hand, are cell surface receptors that play a positive role in cell signaling proliferation. Cd45, a cell surface glycoprotein, plays a critical role in antigen-stimulated dephosphorylation of specific phosphotyrosines that inhibit the Src pathway. [30]

Herstatin Edit

Herstatin is an autoinhibitor of the ErbB family, [31] which binds to RTKs and blocks receptor dimerization and tyrosine phosphorylation. [27] CHO cells transfected with herstatin resulted in reduced receptor oligomerization, clonal growth and receptor tyrosine phosphorylation in response to EGF. [32]

Receptor endocytosis Edit

Activated RTKs can undergo endocytosis resulting in down regulation of the receptor and eventually the signaling cascade. [3] The molecular mechanism involves the engulfing of the RTK by a clathrin-mediated endocytosis, leading to intracellular degradation. [3]

RTKs have become an attractive target for drug therapy due to their implication in a variety of cellular abnormalities such as cancer, degenerative diseases and cardiovascular diseases. The United States Food and Drug Administration (FDA) has approved several anti-cancer drugs caused by activated RTKs. Drugs have been developed to target the extracellular domain or the catalytic domain, thus inhibiting ligand binding, receptor oligomerization. [33] Herceptin, a monoclonal antibody that is capable of binding to the extracellular domain of RTKs, has been used to treat HER2 overexpression in breast cancer. [34]

Small molecule inhibitors and monoclonal antibodies (approved by the US Food and Drug Administration) against RTKs for cancer therapy [3]
Small Molecule Target Disease Approval Year
Imatinib (Gleevec) PDGFR, KIT, Abl, Arg CML, GIST 2001
Gefitinib (Iressa) EGFR Esophageal cancer, Glioma 2003
Erlotinib (Tarceva) EGFR Esophageal cancer, Glioma 2004
Sorafenib (Nexavar) Raf, VEGFR, PDGFR, Flt3, KIT Renal cell carcinoma 2005
Sunitinib (Sutent) KIT, VEGFR, PDGFR, Flt3 Renal cell carcinoma, GIST, Endocrine pancreatic cancer 2006
Dasatinib (Sprycel) Abl, Arg, KIT, PDGFR, Src Imatinib-resistant CML 2007
Nilotinib (Tasigna) Abl, Arg, KIT, PDGFR Imatinib-resistant CML 2007
Lapatinib (Tykerb) EGFR, ErbB2 Mammary carcinoma 2007
Trastuzumab (Herceptin) ErbB2 Mammary carcinoma 1998
Cetuximab (Erbitux) EGFR Colorectal cancer, Head and neck cancer 2004
Bevacizumab (Avastin) VEGF Lung cancer, Colorectal cancer 2004
Panitumumab (Vectibix) EGFR Colorectal cancer 2006

+ Table adapted from "Cell signalling by receptor-tyrosine kinases," by Lemmon and Schlessinger's, 2010. Cell, 141, p. 1117–1134.


Nuclear localization of EGF receptor and its potential new role as a transcription factor

Epidermal growth factor receptor (EGFR) has been detected in the nucleus in many tissues and cell lines. However, the potential functions of nuclear EGFR have largely been overlooked. Here we demonstrate that nuclear EGFR is strongly correlated with highly proliferating activities of tissues. When EGFR was fused to the GAL4 DNA-binding domain, we found that the carboxy terminus of EGFR contained a strong transactivation domain. Moreover, the receptor complex bound and activated AT-rich consensus-sequence-dependent transcription, including the consensus site in cyclin D1 promoter. By using chromatin immunoprecipitation assays, we further demonstrated that nuclear EGFR associated with promoter region of cyclin D1 in vivo. EGFR might therefore function as a transcription factor to activate genes required for highly proliferating activities.


Results

ßTCP binding peptides identified by phage display

Three rounds of panning yielded plaques for three of the six conditions: ßTCP blocked with BSA ßTCP blocked with OBB buffer (non-mammalian blocking buffer) and ßTCP-PLGA composite blocked with OBB buffer. Mock conditions (tubes only) and ßTCP-PLGA blocked with albumin (BSA) did not yield plaques at the 2 nd and 3 rd round respectively. The sequence Leu-Leu-Ala-Asp-Thr-Thr-His-His-Arg-Pro-Trp-Thr was identified in a total of 28% (8/29) of the clones: 5 from ßTCP blocked with BSA 2 from ßTCP blocked with OBB protein buffer and 1 from composite ßTCP-PLGA blocked with OBB buffer, ( Fig 2A ). The remaining 21 clones showed only modest sequence similarity based on the BLOSUM62 scores ( Fig 2A ).

The 12-amino acid consensus sequence (Mw = 1448 Da) includes one negatively charged residue (Asp), one positively-charged residue (Arg), and has a predicted pI of 6.92. Interestingly, the sequence includes two histidines (nominal pK of 6.1), which may become protonated in the low-pH environment of post-surgical inflammation or abstract protons from the calcium phosphate surface. The peptide is predicted to be relatively soluble based on a grand average of hydropathicity (GRAVY) score in the moderately negative range (-0.800). The extinction coefficient (water) at 280 nm was determined to be 5500 M -1 cm -1 .

Binding affinities of EGF fusion proteins with single and concatameric ßTCP binding peptides

Based on the biophysics of interactions between the EGFR and tethered EGF, it is desirable to present the EGF moiety using a spacer to enhance accessibility of the ligand [31,50,51]. We therefore fused the binding peptide sequences to the N-terminus of human EGF (53 amino acids MW = 6.2KDa) with an intervening 106 amino acid sequence comprising a coiled-coil sequence (46 amino acids, MW = 5.4 KDa) flanked on both ends by a flexible, protease-resistant spacer (25 amino acids MW = 1.9KDa) along with several restriction enzyme sites for cloning (See S1 Table for protein sequence). In previous work, we used paired high-affinity heterospecific coiled-coil sequences with the same protease-resistant spacer in order to dimerize EGF and other EGFR family ligands, and had determined that the fusion proteins and their dimers were active when constructed as either N-terminal or C-terminal fusions [48].

Further, we reasoned that the binding affinity of the peptide to ßTCP might be further enhanced by concatamerization of the binding sequence. We used mutagenesis (see Methods) to concatamerize the 12-mer ßTCP binding peptide, yielding protein fusions with 3, 5, and 10 repeats of the 12 amino acid ßTCP binding unit (LLADTTHHRPWT) flanked by other relevant protein domains as depicted in Fig 1 (see Methods). We first examined the relative binding affinities of the fusion proteins as a function of the number of repeats of the binding domain in the fusion protein using a semi-quantitative approach based on eluting proteins followed by western blot analysis ( Fig 2B and 2C ). We titrated the adsorption concentrations across a range of 0� μM and found that binding exhibited a profound dependence on the number of 12-mer repeats (3, 5 or 10) in the binding domain ( Fig 2B and 2C ). Based on these results, we selected the fusion protein with the 10x linear concatamer, referred to as BP10-T-EGF (See Fig 1 ), to perform all subsequent cell interaction experiments.

BP10-T-EGF in solution exhibits wild-type soluble EGF activity

After selecting BP10-T-EGF as the best binder, we confirmed the purity and activity of each recombinantly-produced 10-mer protein batch prior to use in cell phenotypic assays. Western blots of samples subjected to SDS-PAGE showed that the EGF is co-localized with the 73 kDa band ( Fig 3A ), as expected for intact BP10-T-EGF. Biological activity of BP10-T-EGF compared to control wild type EGF was assessed by analyzing activation of Erk-1 and Erk-2 (Erk1/2), a signaling pathway that shows maximal phosphorylation 7� min after stimulation of EGFR in MSC [20,30,31,50�]. Compared to unstimulated controls, a 2.5 to 3-fold increase in ERK1/2 phosphorylation was observed 10 min after stimulation of MSC by either wild type EGF or BP10-T-EGF ( Fig 3B ). Results were normalized to the loading control GAPDH (N = 3 per condition one-way ANOVA pπ.0001 *pairwise comparisons with control were p𢙀.001 using Tukey’s multiple comparisons test all data was log-transformed before analysis). There was no statistical difference between the pairwise comparisons of wild type EGF and soluble BP10-T-EGF (pϠ.05 Tukey’s test). Thus, the EGF domain in BP10-T-EGF appears to be fully competent to activate the EGFR.

Binding and release of BP10-T-EGF tethered to ßTCP scaffolds

Comparable binding isotherms for BP10-T-EGF were observed for crosses of 3 mm and 5 mm using concentrations of 0.2𠄹 μM soluble protein ( Fig 4A ). The resulting range of tEGF surface densities was estimated as 4,000�,000 EGF/μm 2 (see Methods), well within and above the value of 500𠄵,000 EGF/μm 2 found to provide maximal stimulation to epithelial and mesenchymal cells in previous studies employing EGF tethered to polymer substrates [31,50,51]. However, because these previous studies employed tethering schemes that fostered local clustering of tethered EGF, and the binding peptide approach would not necessarily lead to such localized clustering, a tethering concentration of 2 μM (

10,000 EGF/μm 2 ) was chosen for further studies. After a 7-day long incubation of treated (2 μM) scaffolds in 1xPBS at 37C, a

25% release of tethered BP10-T-EGF protein was observed (N = 4 per condition, Fig 4B ). Another stability experiment performed at lower temperatures (4C) using the same buffer revealed there was no statistically significant release of BP10-T-EGF protein from 3mm ßTCP scaffolds (Normalized protein amounts were 1.02 +- 0.09 (day 0) and 1.02 +- 0.11 (day 5) both were normalized to t = 0 N = 3 per condition).

Tethered EGF stimulates an increase in hBMSC number on scaffolds following 7-day culture

After establishing that the EGF domain of the BP10-T-EGF fusion protein induced bioactivity when it was used in soluble form for MSC stimulation (activation of signaling pathways downstream of activated EGFR), we investigated phenotypic responses of low passage primary hBMSC cultured on ßTCP scaffolds modified with BP10-T-EGF fusion protein for three different densities of adsorbed BP10-T-EGF fusion protein. We have previously shown that EGF tethered to polymer substrates via polyethylene oxide (PEO) tethers can enhance proliferation of hBMSC maintained in both expansion and osteogenic media [34] We used day 7 as a metric for comparison in order to allow for several MSC population doublings [54].

Scaffolds (ßTCP crosses, see Methods) were pre-incubated with BP10-T-EGF solutions at concentrations of 0.4 μM, 2 μM, and 9 μM in order to achieve a range of surface densities (estimated as 4,000�,000 BP10-T-EGF per μm 2 ) and to determine dose response. Human BMSCs were seeded onto the treated and control scaffolds and cultured for 7 days in expansion medium. After 7 days, the relative cell numbers were quantified using the Alamar Blue reagent, using cells seeded on standard plates at different densities as a calibration to ensure the assay was in the linear range. All scaffolds treated with BP10-T-EGF had a 2𠄲.3 fold greater number of hBMSC number compared to surfaces without BP10-T-EGF ( Fig 5A N = 3 per condition one-way ANOVA p-valueπ.05 (p-value = 0.02) all pairwise p-values of BP10-T-EGF vs control were π.05 using Tukey’s multiple comparison test). No statistical differences were observed between the different BP10-T-EGF surface densities (All pairwise p-valuesϠ.05 using Tukey’s test). These results indicate that EGF-tethered onto ßTCP scaffolds does not impair expansion of hBMSCs, as the final cell number was greater than the initial number (data not shown), but these data are not sufficient to conclude that tethered EGF enhances proliferation, as differences in initial plating efficiencies together with comparable expansion rates may account for the observed differences at day 7. To parse these mechanisms, we next examined plating efficiencies.

Tethered EGF does not alter plating efficiency of hBMSCs seeded on ßTCP scaffolds

The Alamar Blue assay and other similar kinds of proliferation assays do not have sufficient sensitivity to detect the relatively small number of cells present on scaffolds immediately after seeding. Hence, we developed an approach to directly count cells seeded on scaffolds by embedding scaffolds in agarose, demineralizing to reveal an optically-clear mold, then staining cells and observing them with confocal microscopy. Using this method, we found no statistical differences between the direct count of P3 hBMSCs in the control or BP10-T-EGF conditions for both the 12hr and 24hr time point ( Fig 5B ). This suggests that the increase in hBMSC number observed after a 7-day culture under BP10-T-EGF conditions was most likely due to induction of proliferation and not due to differential plating efficiency.


Where do the phosphate units come from when EGF units dimerize? - Biology

C2006/F2402 '10 Outline for Lecture 24 -- (c) 2010 D. Mowshowitz -- Lecture updated 04/26/10

Handouts From last time: 23C -- Stress Response & TK receptors

New this time: 24A -- Basic Processes in Kidney Tubule
24B -- Kidney Structure

Recent NPR story on oxytocin: When the 'Trust Hormone' is out of balance. http://www.npr.org/templates/story/story.php?storyId=126141922

A. Lactation -- done last time see handout 23A.

B. Stress response (Handout 23C.)

1. Phase one -- Nerve (Sympathetic) activity stimulates target organs (that are not glands) → Direct response of heart, liver, lungs, etc.

2. Phase two -- Nerve (Sympathetic activity) → activation of glands

a. Stimulate pancreas → glucagon release stimulated insulin release inhibited → secretion of glucagon → additional stimulation of some of same target organs

b. Stimulate adrenal medulla → release of epinephrine → stimulation of same targets as sympathetic nerve activity & some additional targets -- hormones can reach where nerves can't go.

3. Phase three -- stimulate HT/AP axis to produce cortisol

HT in brain → releases CRH → AP → releases ACTH → adrenal cortex → produces cortisol → target organs → stimulation of breakdown of fats & protein for energy (sparing glucose for brain) inhibition of immune system.

Note that each additional phase is slower but involves additional degrees of amplification due to second messengers, transcription, etc.

II. Signaling with RTK's. How do RTK's Work?

A. Importance of Catalytic Receptors

  • Many growth factors (such as EGF) and other paracrines, autocrines and juxtacrines act through TK receptors.
  • Insulin, PL, and GH, but not most other endocrines, act through TK receptors. (See Sadava 15.6)

B. Important Properties of Receptor TKs -- See handout 23C & chart below.

1. Receptor is usually a single pass protein

2. Ligand binding usually leads to dimerization of receptors (Sadava fig. 15.6 or Becker 14-17). Why does it matter that TK receptor monomers (or any protein) must dimerize in order to act?

a. Function: If 1/2 the receptors (1/2 the monomers) are abnormal, most of the dimers that form are abnormal.

b. Inheritance: "lack of function" mutations in TK receptors are often dominant. (For an example see Becker figs. 14-20 & 14-21 (14-19 & 14-20). Dimers form, but they are inactive. See below for more details.

  • Example? PLC. Ligand binding to TK-linked receptor can activate a type of PLC → IP3 & DAG → etc.
  • This means that the IP3 pathway can be activated by both types of receptors -- GPCRs and TKs.
  • The cAMP pathway (as far as we know) can only be activated by GPCRs.

c. SH2 domains. Proteins that bind directly to TK have certain types of domains -- usually called SH2 binding domains.

d. Recruitment. Note that the initial target protein(s) to be activated come to or are "recruited by" the TK this is the opposite of the situation with most 2nd messengers where the messenger diffuses throughout the cytoplasm and "seeks out" the target protein to be activated. The recruitment method may be important in localizing the response to a particular part of the cell.

C. A human example of TK receptor signaling: FGF (Fibroblast growth factor) and FGF Receptor. Significance of dimerization. See Becker figs.14-20 & 14-21 (14-19 & 14-20).

1. FGFR is a TK receptor

2. FGF & FGFR needed for proper development as described in Becker chap.14. Failure of signal transmission (or premature transmission) causes developmental abnormalities. See Becker fig. 14-20 (10-20).

3. Achondroplasia (a type of dwarfism), is due to a defective FGF receptor.

4. Achondroplasia is dominant. In a heterozygote for achondroplasia, 1/2 the FGF receptor (monomers) are defective, therefore dimers that form are defective.

a. Example #1: In the example shown in Becker & on handout, the cytoplasmic domain of the defective receptors is missing. Dimers form, but most dimers are never activated -- the two monomers can not phosphorylate each other. (Fig. 14-19) Therefore the signaling is badly disrupted. (In the example shown in Fig. 14-20, the FGF is needed to turn onformation of certain embryonic tissues, so the mutant fails to form these tissues. This type of mutation is called a 'dominant negative' as explained below.)

b. (FYI) Example #2: In most cases of human achondroplasia, the molecular explanation is different. In these cases, the mutation is in the transmembrane domain of FGF3 Receptor and dimers form, but act abnormally. (In these cases, the FGF signal is needed to turn on bone differentiation and turn off cell growth. Mutant dimers signal prematurely, so differentiation starts -- and cell growth stops -- before bones are long enough. This type of mutation is called a 'gain of function' mutation, because it works when it shouldn't.)

5 . Significance of a 'Dominant Negative'

a. Recessive (ordinary) negative mutations. 'Negative' mutations (those that produce inactive protein) are usually NOT dominant. Most negative or "lack of function" alleles (or mutations) are recessive. If there is a mixture of normal and abnormal protein in the heterozygote, the normal, active, protein usually works (in spite of the presence of abnormal, inactive, protein). So usually, overall, there is NO lack of function in the heterozygote.

b. 'Dominant negative' mutations. Sometimes negative mutations are dominant. How does this occur? There is a mixture of normal and abnormal protein present, as usual. What's unusual is that the abnormal, inactive, protein 'gets in the way' and interferes with the working of the normal, active, protein. So overall, there is a lack of function in the heterozygote.

c. Definition: An abnormal allele like the one that produces the FGF receptor without a cytoplasmic domain is called a "dominant negative mutation." A dominant negative allele (or mutation) makes an inactive protein that disrupts function even in the presence of a normal allele (and normal protein). 'Dominant negative' means that the heterozygote is negative for function, not that it doesn't produce any protein.

d. Implications: What does the existence of a dominant negative mutation imply? It indicates that the gene involved probably codes for a protein that must polymerize in order to act.

D. How do GPLR's and RTK's Compare? -- See table below for reference for comparison of basic features of TK or TK linked receptors and G protein linked receptors. For TK's, see Sadava figs. 15.6 & 15. 10 (15.9) or Becker fig. 14-17 for structure and Becker 14-18 for signaling pathway.

# See Becker Fig. 14-4.
## See Becker fig. 14-17 or Sadava 15.6 & 15.3.
*Either part, alpha or beta + gamma may be activator/inhibitor G proteins can also be inhibitory
**SH2 = sarc homology 2 domain

Review problems 6-1 & 6-3

E. Signaling Pathways all interrelate

1. Different 2nd messengers can influence the same enzyme/pathway. See problem 6-20.

2. Each signaling system can affect the others -- For example, Ca ++ levels can affect kinases/phosphatases and phosphorylations can affect Ca ++ transport proteins (& therefore Ca ++ levels). See an advanced text if you are interested in the details.

3. The same signal (same activated TK receptor) may trigger more than one signaling pathway . For example, EGF can trigger both the IP3 pathway and the ras pathway. (Becker fig. 14-18).


IV. Intro to Kidney Function (Handout 24A). See also Sadava Sect. 51.4. & 51.5.

Here's an article from the LA Times on a recent artificial kidney.

A. Overall Function -- what does the kidney do?

  • Filtration

  • tubular secretion (addition of substances to the filtrate) = secretion into the tubule

  • tubular reabsorption (removal of substances from filtrate) = reabsorption from the tubule

  • control of volume of urine

B. Details of Basic Processes

1 . Basic set up

a. Capillaries: Artery (from heart) → afferent arteriole → glomerular capillaries → efferent arteriole → peritubular capillaries → venule → vein (back to heart)

b. Filtration: Material moves from glomerular capillaries into tubule.

c. Secretion & Reabsorption: Materials moves between inside of tubule and inside of peritubular capillaries (surrounding kidney tubule).

2. The 4 Basic Processes

a. Filtration:

(1). Occurs in glomerulus

(2). About 20% of blood liquid (plasma) enters Bowman's capsule = filtrate

(3). Filtrate contains no large proteins or cells

b. Tubular (selective) secretion: Material is added to the filtrate.

  • Secretion = extruded by the cells into extracellular space (into filtrate, lumen, etc.).

  • Excretion = carried out of body in urine or feces.

c. Tubular (selective) reabsorption: Material is removed fromthe filtrate.

(1). Result of reabsorption: Filtrate does NOT carry certain materials (which are selectively reabsorbed) -- conserves valuable materials returns them to circulation.

(2). Aldosterone affects Na + reabsorption (& K + secretion). Details below.

(1). Water loss is adjusted at the end of the tubule using ADH. (See below.)

(2). Urine can be more -- or less -- concentrated than the plasma. Concentration and/or volume can be varied to suit need.

(3). Water loss or conservation in tubule controls volume of body fluids (not just urine volume). Controls volume of plasma, extra cellular fluid, etc. (& blood pressure).

3. How does tubular secretion/reabsorption occur? Structure of cells lining tubules -- see handout 24A bottom or Sadava fig. 51.12 (for a different example).

a. Tubules are lined by layer of polarized epithelial cells (similar to those lining intestine)

b. Materials must cross epithelial cells to enter or exit lumen of tubules.

c. Interstitial fluid separates epithelial cells and peritubular capillaries.

d. Epithelial cells have different proteins/channels/transporters on their two surfaces -- the apical or luminal surface (facing lumen) and basolateral surface (facing interstitial fluid and capillaries).

e. Cells in different parts of the tubule have different transporters/channels on their luminal surface.

f. All cells in tubule that absorb Na + have the Na + /K + pump on their basolateral surface. Other transporters may vary.

g. What cells transport (& in which direction) depends primarily on which transport proteins are on the luminal surface. Depending on transporters, cells can secrete materials into lumen or reabsorb material from lumen.

h. Cells lining tubule do actual secretion/reabsorption but peritubular capillaries remove reabsorbed material or provide material to be secreted. Therefore (as shown on handout 24A, top left):

(1). Result of tubular reabsorption = net transfer from filtrate to capillary.

(2). Result of tubular secretion = net transfer from capillary to filtrate.

Try problem 12-3.

3. Example of reabsorption -- see 24A upper right. (Fig. 14-18). How Na + is reabsorbed.

Q: How could K + be secreted? What would you have to add/remove from the diagram?

  • aldosterone affects Na + reabsorption (& K + secretion)

  • ADH affects water reabsorption

b. Role of aldosterone in Na + reabsorption

(1). Promotes reabsorption of Na +

(2). Stimulates virtually all steps of reabsorption -- all steps shown in 24A, upper right.

c. Role/Mech. of action of ADH

  • Are they in the luminal membrane, BL membrane, or both?

  • Are they inserted or removed in response to hormone?

  • Why are cells in only some areas of tubule responsive to ADH (or aldosterone)?

d. Questions to think about: Where are the receptors for ADH? Aldosterone? Which hormone elicits a faster response?

See problems 12-8 to 12-10.(See below for location of cells affected by each hormone.)

A. Overall structure -- see handout 24B or Sadava fig. 51.9. Alternatively, see Kimball's biology pages.

1. Kidney has medulla (inner part) and cortex (outer)

2. Functional unit = nephron (Sadava 51.7 )

3. Visible unit (in medulla) = Renal Pyramid = bottoms of many nephrons

4. Tops of nephrons in cortex

B. Structure of Nephron -- see handout 24B or Sadava fig. 51.7 & 51.9 . For EM pictures see Sadava 51.8. (We may do the parts as we need them, but all are summarized here.)

1. Nephron itself -- parts in cortex

a. Bowman's capsule
b. proximal (convoluted) tubule
c. distal (convoluted) tubule

a. Loop of Henle
b. Collecting duct (shared by many nephrons)

3. Capillaries (discussed last time)

a. 2 sets in series

(1). Glomerular
(a). form glomerulus inside Bowman's capsule
(b). function in filtration

(2). Peritubular
(a). surround tubules
(b). fyi: part in medulla (surrounding loop of Henle) is called the vasa recta
(c). function in secretion & reabsorption

b. How capillaries connected. Circulation goes as follows:

Artery (from heart) → afferent arteriole → glomerular capillaries → efferent arteriole → peritubular capillaries → venule → vein (back to heart)


V I. Kidney Function, revisited.

A. Function of Nephron -- Let's follow some liquid through.

2. Reabsorption & secretion (of most substances) occurs in proximal tubule

3. Loop of Henley -- overall picture of state of filtrate

a. Definition: Osmolarity (Osm) = total solute concentration = concentration of dissolved particles = osmol/liter. (One osmol = 1 mole of solute particles.)

Examples: 1M solution of glucose = 1 Osm 1M solution of NaCl = 2 Osm.

(1). Descending: Osmolarity increases as filtrate descends due to loss of water

(2). Ascending: Osmolarity decreases as filtrate ascends due to loss of salt reaches min. value less than that of blood. Therefore can excrete urine that is hypo-osmotic (less concentrated) than blood.

(3). Overall: Net effect of going through countercurrent loop -- less volume, less total salt to excrete (even if filtrate and blood are iso-osmotic when done).

4. Distal Tubule & Collecting Ducts

a. Control of removal from filtrate (reabsorption) of remaining Na + (Role of aldosterone.)

b. Volume Control -- occurs in collecting ducts. (Role of ADH.)

B. Details for Proximal Tubule

1. Many substances removed from lumen by secondary act. transport

a. examples: glucose and amino acids

b. AA etc. cross apical/luminal surface of epithelial cell by Na + co-transport -- therefore a lot of Na + removed from filtrate (along with glucose, AA, etc.)

c. Exit basolateral side of cells into intersit. fluid by facilitated diffusion

c. Process is similar to absorption in cells lining intestine

2. Na+/K+ pump on basolateral side keeps internal Na+ low.

4. Secretion of most materials (except K + ) occurs here -- toxins etc. transported to filtrate

C. Details of Transport Events in Loop of Henley (See Sadava 51.10)

1. Water permeability. Luminal cell membranes in descending loop and lower part of ascending loop are permeable to water.

2. Generating the Na + gradient in the medulla.

a. Luminal cell membranes in rest of ascending are impermeable to water and pump NaCl from lumen to interstitial fluid.

b. NaCl pumped out from ascending loop accumulates in medulla, forming a gradient of increasing osmolarity (outside the tubule) as reach bottom of loop = core of medulla.

3. Water loss: Filtrate from proximal tubule loses water as it descends into medulla → NaCl stays in tubule → high concentration NaCl in tubule → to be removed in ascending. (Na + not pumped out of these cells on BL side.)

4. Escalator Effect: If NaCl diffuses into descending loop, it is carried around and pumped out in ascending = escalator effect.

5. Why called countercurrent? Because flow in two sides of loop is in opposite directions -- physically and with respect to osmolarity.

a. First leg (descending) of loop removes water → higher osmolarity in filtrate as it proceeds (on the way down).

b. Second leg (ascending) removes salt → lower osmolarity in filtrate as it proceeds (on the way up).

c. Net effect is higher osmolarity toward the bottom on both legs.

d. Why doesn't flow in peritubular capillaries (vasa recta) wash out the salt gradient in the medulla? Because capillaries exit out the top of the nephron, carrying a low amount of salt.

See problems 12-1 to 12-3.

D . Distal Tubule and Collecting Ducts -- A few more Details

1. Overall

a. Reminder: Filtrate entering distal tubule is at minimum osmolarity

b. This is the only part of the tubule affected by ADH and aldosterone

(1). Events in distal convoluted tubule (& first part of coll. ducts) depend on aldosterone

(2). Events in collecting duct (volume control) depend on ADH

c. Hormones cause water and/or remaining Na + to be removed (reabsorbed from filtrate)

(1). aldosterone affects Na + reabsorption (& K + secretion) -- See handout 24B, top right.

(2). ADH affects water reabsorption

2. Importance of aldosterone (in water/Na+ balance)

a. Promotes reabsorption of Na + water follows (not necessarily in same part of tubule).

b. Amount of Na + reabsorbed due to aldosterone is small % of total, but adds up affects blood pressure.

c. Aldosterone promotes K + secretion -- this may be of major importance, but we are focusing on role of hormone in Na + balance.

3. Importance of ADH. Controls water retention in body. Osmolarity of filtrate will increase (and volume decrease) in collecting duct if ADH (vasopressin) present and water removed. (See above for mechanism.)

4. Where do the hormones come from? What triggers their production/release?

a. ADH produced by HT (& released in PP) primarily in response to high osmolarity of blood.

b. Aldosterone produced by adrenal cortex in response to inadequate blood flow through kidney, not primarily in response to ACTH.

See problems 12-8 to 12-13 & 12-15.

Next Time: Last Lecture! How Kidney Function & Blood Pressure are Regulated Cancer & Control of Cell Growth


Regulation of Cell Proliferation by Receptor Tyrosine Protein Kinases

Cross-linking of receptors causes activation

Tyrosine kinase -containing receptors come in several different forms but they are unified by the presence of a single membrane-spanning domain and an intracellular kinase catalytic domain. The extracellular chains vary considerably as indicated in Figure 10-3 . A general feature of these receptors is that ligand binding causes dimerization, first discovered for the EGF receptor ( Yarden and Schlessinger, 1987 Sternberg and Gullick, 1990 ). In addition to activation by the natural peptide ligands, some (but not all) of the functions of the EGF receptor can be elicited by crosslinking with antibodies ( Defize et al., 1986 Spaargaren et al., 1991 ). Within the family of the receptor tyrosine kinases, ligand-mediated crosslinking is achieved in a number of ways. Platelet-derived growth factor (PDGF) is itself a disulfide-linked dimeric ligand which cross-links two receptors upon binding. FGF uses a heparin oligosaccharide to bring two receptors together. In the case of the insulin receptors, they are already dimerized through a disulfide bridge but nevertheless require insulin in order to signal into the cell ( Ottensmeyer et al., 2000 Yip and Ottensmeyer, 2003 ). EGF is different, its binding causes the receptor to unfold, exposing a dimerization motif that allows the occupied monomers to recognize each other (or to recognize unliganded unfolded ERBB2) ( Yamanaka et al., 2002 Gerrett et al., 2002 ) ( Figure 10-5 ). Since truncated EGF receptors that lack the extracellular ligand-binding domain are constitutively active ( Thiel and Carpenter, 2007 ), it follows that the unoccupied extracellular domain acts to prevent kinase activation. Indeed, dimerization of the receptor brings about a change in the relative position of the transmembrane domains that, in turn, promotes dimerization and activation of the intracellular protein kinase segment ( Endres et al., 2013 ).

Figure 10-5 . Dimerization of the EGF-receptor extracellular segment. (a) The EGF receptor is composed of four extracellular domains, L1, CR, L2, and GH. L1 and L2 carry leucine-rich repeats that function in ligand binding, CR1 is a furin-like cysteine-rich domain and GH is a growth factor receptor domain IV. The intracellular segment contains a tyrosine kinase domain and a long unstructured C-terminal sequence that harbors numerous phosphorylation sites. The C-terminal tail is the substrate of a neighboring receptor (phosphorylation in trans). Note that in the receptor structure on the left, the extracellular segment folds onto itself the dimerization arm of the CR domain (indicated by a green surface) docks onto the GF domain. The receptor shown is bound to EGF but only to the L1 domain. (b) Binding of EGF to both L1 and L2 causes unfolding and dimerization of the extracellular segment. The dimerization arm of CR now binds to a docking site in the CR domain of the partner (and vice versa). As a consequence, the two transmembrane domains associate with their N-terminal sequences and this, in turn, brings about a change in the position of the kinase domains leading to their activation (not shown, see Figure 10-6 ).

Normally, the kinase domain of the EGFR is inactive because both the activation segment and the αC- helix are wrongly positioned. Moreover, the kinase is tethered to the plasma membrane through both its N-terminal juxtamembrane sequence, which three leucines (motif LLRRL), and through interactions of surface lysines and arginines (positively charged) with negatively charged phospholipids ( Figure 10-6 ) ( Arkhipov et al., 2013 ). Membrane-tethered kinases cannot form dimers. Receptor crosslinking disrupts the membrane binding and allows the two kinases to align asymmetrically, with the C-lobe (activator kinase) binding to the N-lobe (receiver). This causes an outward movement of the activation segment and inward movement of the αC-helix of the receiver kinase ( Figure 10-6 ). The other, activator kinase, remains catalytically incompetent. The EGFR is rendered competent through an allosteric interaction between kinase domains (in other words, an activating “shape change” is caused by protein–protein interaction) ( Jura et al., 2009 Brewer et al., 2009 ). The interaction between the two EGFR-kinase domains is reinforced by the antiparallel association of the helices carrying the –LRRLL motif and by the juxtamembrane region of the receiver kinase which latches onto the activator (juxtamembrane latch). The mode of activation is unique among the tyrosine protein kinases, which otherwise often involves phosphorylation of the activation segment (further discussed in Chapter 15, “Activating the Adaptive Immune System: Role off Non-receptor Tyrosine Kinases” and Chapter 16, “Signaling through the Insulin Receptor” ). However, the mechanism is similar to the one observed with cyclin-dependent protein kinases (CDKs), which are serine/threonine protein kinases. Here allosteric regulation is brought about by a regulatory subunit, named cyclin, that binds the kinase domain.

Figure 10-6 . Allosteric regulation of the EGFR kinase domains through the formation of an asymmetric kinase dimer. In the inactive state (monomer) the kinase domain is tethered to the membrane through an interaction of the LRRLL motif in the juxtamembrane sequence and through binding of lysine residues of the kinase domain to negatively charged lipids. Ligand-mediated dimerization of the extracellular segment leads to dimerization of the transmembrane helix and this disrupts membrane attachment of the kinase domain. Two protein kinase domains now associate head-to-tail (asymmetric dimer) bringing about a conformational change of one kinase only, named the “receiver kinase.” As a consequence, the multiple residues on the adjacent C-terminal tail are phosphorylated. As the kinase domain may change roles, where the activator becomes the receiver kinase, with time both tails will be fully phosphorylated. The phosphate symbol indicates a phosphorylation site (threonine-678) in the juxtamembrane region which inactivates the receptor (part of a negative feedback mechanism).The residues of the human EGFR are numbered according to the sequence information in UniProtKB (with inclusion of the signal peptides, 1–24).

It is likely that the receiver and activator positions can be interchanged, so that the kinases alternate their activity. Indeed EM studies of EGF-treated receptors have revealed that the dimeric extracellular segment is associated with intracellular segments of different conformations, ranging from asymmetric dimers (active), symmetric dimers (inactive) to nonassociated kinase domains ( Mi et al., 2011 ). Importantly, as a consequence of asymmetric dimerization, the C-terminal tails of the two kinases are trans-phosphorylated (one kinase phosphorylates the tail of the other) on multiple tyrosine residues. The phosphorylated dimer then constitutes the active receptor. For more information about protein kinase activation, return to Chapter 2, “An Introduction to Signal Transduction,” Section “Protein kinase activation mechanisms” ( Figures 2-43 and 2-44 Figure 2-43 Figure 2-44 ).

While certain proteins serve as substrates of the activated EGF receptors, what really matters is the recruitment of signaling partners through the phosphotyrosine residues in the C terminal tail. These phosphotyrosines, within a specific context of five amino acids, bind proteins bearing SH2 or PTB domains ( Liu et al., 2010 ). The recruited proteins can be effectors or adaptors, which serve to bind effectors indirectly ( Anderson et al., 1990 Jones et al., 2006 ) (see Figure 10-4 for a list of such effectors and adaptors, and see Figure 10-8 for more detail about the interaction).

Growth factor receptor dimers can further aggregate into oligomers of several hundreds or even thousands of units. This phenomenon, already recorded at the time when receptor dimerization first came to light ( Yarden and Schlessinger, 1987 ), has now been visualized by the use of fluorescent protein tagging ( Carter and Sorkin, 2006 ). Different types of receptors can be recruited, so that PDGF receptors have been found associated with EGF receptors ( Saito et al., 2001 ) and these aggregates give rise to multiprotein signaling platforms that may contain numerous effectors.


Lectins are carbohydrate-binding proteins. Individual lectins show specificity for particular sugar structures.

This is a homology unit of ∼ 110 amino acids. Although there is variation in primary sequence, the structure of this domain is conserved and organized into two β-sheets, one of three and the other of four strands, which are stabilized by disulphide bonds in most of the cases. Ig domains are found in several proteins, including cell-adhesion molecules and signalling receptors, and have been implicated in protein–protein interactions.

Fibronectin type III (FNIII) domain

FNIII motifs, originally described in the extracellular matrix protein fibronectin, comprise ∼ 90 amino acids. Their three-dimensional structure is similar to that of the immunoglobulin domain. In fibronectin itself, a RGD sequence in the loop that connects the β-sheets in FNIII motif 10 has been implicated in promoting adhesion by binding to integrins. This motif is also found in a wide variety of signalling proteins.

The pKα (also known as pKa) is a measure of the uptake/release of protons by amino acids. It is the negative log to the base 10 of the acid-dissociation constant, which reflects the equilibrium between protonation and deprotonation and indicates the extent of proton dissociation. The log scale is used because this constant differs over orders of magnitude between individual acids the smaller the pKα value, the stronger the acid.


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