Receptor Tyrosine Kinases: Shaw lecture 2: 11/20/99
There are two major classes of cell surface receptors which we will concentrate on in this course. These are the receptor tyrosine kinases which we will discuss here and the G protein coupled receptors.
Families of receptor tyrosine kinase
Most of these growth factor receptors are single membrane spanning molecules with a tyrosine kinase in the cytoplasm. These typically have some combination of IgG, fibronectin type III, EGF repeats etc. in the extracellular domain. There are several distinct families or receptor tyrosine kinases, each of which generally have related ligands, similar catalytic and similar extracellular domains. For example all of the related neurotrophin molecules (NGF, BDNF, NT3 and NT4/5) bind to the related Trk receptors (trkA, TrkB and TkrC). Presumably each family of ligands and receptors arose from one ligand/one receptor pairs which then each went through cycles of gene duplication.
List of these families
1. Platelet derived growth factor (PDGF) family, includes colony stimulating factor 1, (CSF-1), Vascular endothelial derived growth factor (VEGF) and others. These all have an insert in the kinase domain of ~80 amino acids which contains several tyrosine residues which can be phosphorylated and bind to a subset of SH2 containing proteins.
2. Fibroblast growth factor (FGF) receptor family, bind the FGF growth factors which are involved in angiogenesis, mesoderm induction in embryogenesis among many other processes.
3. Insulin receptor family, including the insulin and insulin like growth factor-1. These are composed of four polypeptide chains, two of which are entirely extracellular and disulfide bridged to each other and to two identical subunits with membrane spanning and tyrosine kinase domains.
4. Epidermal growth factor (EGF), Oncogenic when overexpressed, also found as transforming oncogene in tumor viruses under various names, Neu, erbB etc.
5. Trk receptors, a family including trkA, trkB and trkC. These are receptors for the neurotrophins, a potent family of neuronal growth factors including NGF, BDNF, NT3, NT 4/5.
6. Hepatocyte growth factor family (HGF). MET was characterized as a potential oncogene, but is the normal unmutated HGF receptor. Simply overexpressing this receptor tends to make a cell cancerous.
7. EPH family is the largest family with more than 20 members described to date. These are heavily expressed in the nervous system. Their ligands are the ephrin family of proteins, which can be divided into two class Ephrin A (GPI anchored to the membrane) and B (transmembrane proteins). EPH family receptors and their ligands are implicated in mediation of developmental events particularly in the nervous system.
8. UFO/Axl/Tyro-3: these are of unknown function and the ligand is unknown. Found in the nervous system. Overexpression of mRNA is enough to lead to cancer.
How they work
These molecules typically exist as inactive monomers in the plasma membrane in the absence of ligand. Ligand binding crosslinks such monomers to form dimers and allows each kinase domain to phosphorylate the other, activating both kinases and possibly then going on to phosphorylate other proteins. In many cases (e.g. the epidermal growth factor receptor), the kinases phosphorylate their partners in the activation loop thus increasing their activity. Most of the ligands, such as PDGF or the neurotrophins are dimers, thus allowing the receptors to dimerize. This is a very simple and elegant method allowing the ligand to generate a signal inside the cell. Compare this mechanism with that used by G protein coupled receptors, in which a conformational change is transmitted across the seven transmembrane a-helices into the cell.
Tyrosine kinases autophosphorylate on a variety of sites, generally in regions flanking the kinase domain or on inserts in the domain. These sites are then available for binding to specific subsets of SH2 domain and Ptb domain containing proteins, e.g. enzymes such as PLC-g, PI-3 kinase, Src family kinases, regulators of small G proteins, adapter proteins. The formation of these various protein complexes generally brings the enzyme into contact with its substrate; lipid modifying enzymes like PLC-g and PI-3 kinase are brought into contact with lipid, which is their substrate. Small G proteins are almost all membrane localized by C-terminal farnesylation or geranylgeranylation, and the guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) must also be membrane localized in order to act on them. The autophosphorylated receptor can also go on to phosphorylate other proteins. Phosphorylation on tyrosine residues may affect the activity of an enzyme but may also have another consequence, to allow the phosphorylated protein to bind to yet other proteins by means of SH2 domain interactions.
This elegant system of signal transduction described in outline above is unfortunately rather prone to problems. Gene and chromosome amplification resulting in over-expression of the receptor can be associated with cancer in the case of for example HGF and EGF receptors. A particularly interesting example of a tumor association is the Trk Receptor. Trk later known as trkA was discovered in gene transfer experiments using DNA from a human colon cancer. The original trk protein is the result of a gene fusion between the tropomyosin gene and what at the time (1986) was an unknown receptor tyrosine kinase, hence the name, tropomyosin related kinase (trk). Tropomyosin is an abundant cytoskeletal protein which has an extremely strong tendency to dimerize. The effect of adding this molecule to the N-terminus of a receptor tyrosine kinase is bad for two reasons. Firstly it generates a large amount of protein, since the tropomyosin promoter is a strong one, and receptor tyrosine kinases are normally not very abundant. Secondly the tyrosine kinase domains are held together constitutively, so that they are always activated. The normal tyrosine kinase which formed part of this gene fusion was cloned out and by about 1991 it had become clear that it was the receptor for the prototype nervous system growth factor, nerve growth factor (NGF).
There are several variants on the simple scheme discussed above.
Insulin receptor signaling
More complex is the insulin receptor. In this case the receptor already exists as a tetramer, and the activated complex phosphorylates itself and two other proteins IRS-1 and IRS-2 (IRS = insulin receptor substrate). These proteins have an N-terminal PH (pleckstrin homology) domain and a Ptb (protein tyrosine binding) domain (a phosphotyrosine binding site distinctly different from the SH2 domain, but structurally similar to PH domains) and ~20 sites of correct consensus sequence for tyrosine phosphorylation with the consensus YMXM and YXXM. Several of these bind to p85 regulatory subunit of PI3 kinase. Can also bind SH-PTP2 (a.k.a. PTPII, Syp, a tyrosine phosphatase) Nck, Shc, Grb2/mSOS. In the case of the insulin receptor most of the SH2 domain containing proteins bind to the IRS-1 adapter rather than the receptor itself, which is presumably an amplification mechanism.
B and T lymphocyte receptors
The IgM (in B cells) and the T cell receptor are associated with various types of transmembrane subunits almost all of which contain immunoreceptor tyrosine based activation motifs (ITAMs), also known as antigen receptor homology 1 regions (ARH1). ITAMs are of the sequence D/E,X(7-8),D/E,XXYXX,L/I,X7YXX,L/I. Both Y (tyrosine) residues can be phosphorylated by non-receptor tyrosine kinases of the Src family in activated cells. When antigen is present the Src kinases are dephosphorylated, possibly by the cell surface tyrosine phosphatase CD45. The activated Src family kinases somehow recognize clumped receptor complexes, which occurs when antigen is around. The src family kinases phosphorylate subunits of the B and T cell receptor complexes, including the two in the ITAM/ARH1 sequences. The phosphotyrosine residues recruit the SH2 domain containing non-receptor tyrosine kinase Syk (in B cells) and Zap-70 in (T cells), which both have two SH2 domains and apparently bind to the two tyrosine phosphates in the ITAM/ARH1, and are then activated and tyrosine phosphorylate down stream targets, including IRS-1, IRS-2 etc. Since IRS-1 had up to 20 SH2 domain binding sites, considerable amplification can take place.
Tyrosine kinase associated receptors
Lymphokines/cytokine receptors; receptors for growth hormone, Prolactin, leukemia inhibitory factor, erythropoetin, Ciliary neurotrophic factor (CNTF), interleukins, interferon, leptin receptor and many others. These are transmembrane receptors which do not contain an intrinsic tyrosine kinase activity, but nonetheless function in a manner very similar to the receptor tyrosine kinases. Multiple subunits which come together in various combinations. Many of the receptors contain so-called box1 and box2 sequences allow binding of ~130kDa protein tyrosine kinases of the JAK family of non-receptor tyrosine kinase, of which there are at least 4 examples (JAK1, JAK2, JAK3, TYK2). JAK stands for janus kinase (the two faced roman god), since these molecules appear to have two kinase domains. (Alternately I've heard Jak was derived from "just another kinase"). JAKs are large kinases with what appear to be two tyrosine kinase domains. Basically, like with the receptor tyrosine kinases, the receptors exist as either monomers or dimers in the absense of ligand but aggregate when ligand is around, and aggregation results in autophosphorylation and activation of the JAKs and also phosphorylation of the receptors. One of the notable substrates of activated JAKs is the STAT family of proteins (STAT = signal transducer and activator of transcription). These bind to the phosphorylated receptor by means of their SH2 domains, and are phosphorylated by JAKs. One STAT then binds to another STAT, by means of an SH2 domain-Phosphotyrosine interaction, and this dimeric form migrates to the nucleus where it binds to specific DNA response elements, hence helping in the activation of specific lymphokine/cytokine induced genes. This is one of the most direct pathways by which a ligand can direct gene expression in the nucleus.
Glial cell line derived neurotrophic factor (GDNF) and Neurturin receptors.
Yet another variant on the general theme is provided by the receptor system for GDNF and Neurturin. These are two related and important neuronal growth factors which maintain various kinds of neurons, and other members of this growth factor family have been discovered. The receptors for these factors are totally extracellular and are anchored to the membrane by a GPI anchor. The extracellular receptors for GDNF and Neurturin, called GDNFRa and NTRa , are related to oneanother but are distinct and bind specifically only to GDNF or Neurturin. However both activate the same transmembrane tyrosine kinase, the Ret proto-oncogene.
Receptor tyrosine phosphatases.
The tyrosine kinases are only useful as signaling molecules because the phosphate groups they add can be rapidly and controllably removed. This is performed by specific tyrosine phosphatases. These are very numerous but are generally less well understood than the tyrosine kinases. It is estimated that the human genome may contain 500 different tyrosine phosphatases, about the same as the estimated number of tyrosine kinases. There are therefore far more tyrosine phosphatases than serine/threonine phosphatases, and this may explain why most of the phosphate on proteins is on serine and threonine residues. Like the kinases, the phosphatases can be divided into membrane spanning and cytoplasmic superfamiles. The membrane spanning phosphatases typically contain IgG repeats, fibronectin type III repeats and other domains typical of adhesion molecules. Transfection of some of these molecules results in causing cells to clump, so that they probably have a role in cellular adhesion, and perhaps some of them mediate contact inhibition. This is the well known phenomena by which normal cells in tissue culture grow to form a monolayer and then stop dividing. Perhaps activation of tyrosine phosphatases inhibits growth factor mediated signaling and hence prevents cell division. For most of these molecules the ligands are not known and they are generally much less well understood than the tyrosine kinases.