A comparison of the endodomains of representative members of six of the sub-families of the human RTK superfamily is illustrated in . In panel A, these are shown in linear fashion, aligned on the boundaries of the kinase domains, with the full complement of tyrosine residues of each one indicated. Those residues known to be phosphorylated are indicated in red and the aligned activation loop regions are highlighted.
Figure 1 Comparison of six different RTK endodomains, illustrating the diversity in organization and molecular signaling mechanisms. (A) Linear plot showing the location of the tyrosine residues; those that have been shown to be phosphorylated (according to UniprotKB (more ...)
This projection readily demonstrates that while the size of the kinase domain is relatively constant (albeit some members of the superfamily do have kinase inserts, most notably the platelet-derived growth factor receptor (PDGFR)), the size of the two regions that straddle the kinase domain can be significantly different in length, making the overall size of the intracellular domains distinct. For example, TrkA contains only 356 residues while PDGFRβ has 552 amino acids; the others depicted falling in between ().
Comparison of cytoplasmic domains of RTKs.
The residues comprising the JM and CT regions are dramatically different. Both the PDGF and epidermal growth factor (EGF) receptors have large CT domains (144 and 231 residues, respectively) while the TrkA and the discoidin domain receptors (DDR) have only 15 and 8 amino acids, respectively. In contrast, DDR1 (isoform1) has the largest JM region in the RTK superfamily: 171 residues.
The total number of tyrosines found in each endodomain and those that have been shown to be modified are substantially different () and their distribution (both modified and unmodified) is quite variable (See ). For example, the PDGFRβ endodomain contains 27 tyrosines, while the TrkA endodomain has only 11. Quite clearly only a subset of the total tyrosines present in any given receptor are actually converted to their phosphorylated derivatives following stimulation and, of those converted, only a subset actually play an active role in signaling. These differences are depicted schematically in , where the positions of only the tyrosines known to be involved in downstream signaling are shown. These assignments are based for the most part on direct identification of the germane phosphorylated peptide by proteomic techniques. In some cases they are based on comparisons among similar family members. One modification that is commonly found in the RTK superfamily is the phosphorylation of a pair of tyrosines located closely adjacent to each other in the activation loop (). In many cases a third tyrosine located a few residues upstream is also included in this group. The pair of phosphotyrosines are required for function and mutation of one or both inactivates the kinase domain. Only EGFR of the commonly studied members of the superfamily is an exception to this rule. It has only a single tyrosine in this position and although it is phosphorylated on activation by EGF it is not required for kinase function. The role of the activation loop tyrosines is to stabilize the loop in an open conformation so that both ATP and the substrate peptide can be bound(Hubbard and Till, 2000
The function of the other essential phosphotyrosines is to provide binding sites for the soluble (or membrane-anchored) proteins that are recruited following receptor activation(Pawson, 2002
). These include scaffolding proteins, which provide single or multiple sites for additional docking interactions and individual effectors. In some cases more than one docking protein is known to interact with a site, e.g. Shc and FRS2 with Y490 in TrkA(Kaplan and Miller, 2000
); in others the same entity may bind to more than one site on the receptor, e.g. Gab1 to Y1068 and 1086 of the EGFR(Liu and Rohrschneider, 2002
). Not surprisingly, given the substantial differences in organization of the RTK endodomains, these interactions are quite different. The insulin and FGF receptors mainly function by binding large scaffolding proteins that are themselves tyrosine phosphorylated and subsequently dock multiple adaptors/effectors to amplify and propagate the signal. In some cells, TrkA can also utilize the FRS scaffold that is utilized by FGFR. Interestingly, this interaction requires that Y490 be modified, while in FGFR, the interaction is constitutive (no phosphorylated tyrosine is required)(Ong et al., 2000
). Indeed, FGFR1 has only a single phosphotyrosine docking site, located at the border between the kinase and C-terminal domain; all of the remaining tyrosines (including some that can become phosphorylated) are not required for signal transduction as measured in a PC12 cell assay(Foehr et al., 2001
). In contrast, the PDGF and EGF receptors have multiple sites that are modified and utilized. Although some are more significant than others, large deletions or multiple substitutions still leave the receptor able to signal, albeit with less potency. For example, EGFR mutants with the 5 principal tyrosine sites in the CT domain converted to phenylalanine or with the excision of the entire CT domain can still activate the Ras/ERK pathway in PC12 cells(Tyson et al., 2003
) or NIH 3T3 cells(Gotoh et al., 1994
). This multiplicity makes dissection of the roles of individual sites within these receptors difficult.
Both Trk and DDR1 present a more simplified structure. In TrkA, there are only two tyrosine docking sites that have been defined and mutation of these sites gives measureable changes in various assays. Thus, TrkA is an excellent candidate for further analysis of downstream changes in the PTM profile generated by NGF, its ligand. DDR1 also seems to have a relatively simple signaling structure, but this has not yet been defined as clearly as TrkA (Vogel et al., 2006
RTKs are known to activate several pathways that are linked to cellular phenotypic responses that have been extensively described(Schlessinger, 2000
). Primarily these involve the activation of the ERKs, usually via Ras and a cascade of subsequent kinase activations; phospholipase Cγ, which cleaves phosphoinositides to produce diacylglycerol and inositol trisphosphate; and the Akt-linked pathways, which are initiated by the activation of phosphoinositide-3-kinase (PI-3-K). While the initiating events that immediately follow the receptor activation extensively (if not exclusively) involve tyrosine phosphorylation, the longer term propagation of the signal generates (and certainly requires) a much greater number of serine/threonine modifications. In most cellular paradigms, by approximately 20 minutes, the majority of the phosphotyrosine modifications have been replaced by a much larger number of serine and threonine phosphorylations, along with the expansion of Nε
-acetylation, ubiquitination and O-GlcNAc modifications(Choudhary and Mann, 2010
). All of these modifications are reversible in nature and specific enzymes to remove these PTMs are well known. However, it has been shown by numerous proteomic experiments that the there is a substantial basal level of all of these modifications, necessitating careful quantitative measurements to detect those PTMs that have significantly changed as a result of receptor stimulation. It is those entities with increased (or decreased) stoichiometries of modification that are eventually instrumental in producing the cellular/phenotypic responses driven in large part by transcriptional modulation. This provides many potential targets for therapeutic/diagnostic applications, some of which have already been exploited.