Discoidin domain receptor 1 (DDR1) signaling in PC12 cells: activation of juxtamembrane domains in PDGFR/DDR/TrkA chimeric receptors

The discoidin domain receptor (DDR1) is characterized by a discoidin I moti f in the extracellular domain, an unusually long cytoplasmic juxtamembrane (JM) region, and a kinase domain that is 45% identical to that of the NGF r eceptor, TrkA. DDR1 also has a major splice form, which has a 37 amino acid insert in the JM region with a consensus Shc PTB site that is lacking in t he shorter receptor. One class of ligands for the DDR receptors has recentl y been identified as being derived from the collagen family, but neither na tive PC12 cells, which express modest amounts of DDR1, nor transfected PC12 cells, which express much larger amounts of DDR1, respond to this ligand. A chimeric receptor, containing the extracellular domain of hPDGFR beta fus ed to the transmembrane and intracellular regions of DDR1, also fails to me diate neuronal-like differentiation in stably transfected PC12 cells and is only weakly autophosphorylated. However, chimeric receptors, which are com posed of combinations of intracellular regions from DDR1 and TrkA (with the extracellular domain of hPDGFR beta), in some cases provided ligand (PDGF) -inducible receptor responses. Those with the TrkA kinase domain and the D DR1 JM regions were able to produce differentiation to varying degrees, whe reas the opposite combination did not. Analysis of the signaling responses of the two chimeras with DDR1 JM sequences (with and without the insert) in dicated that the shorter sequence bound and activated FRS2 whereas the inse rt-containing form activated Shc instead. Both activated PLC gamma through the carboxyl-terminal tyrosine of the TrkA domain (Y785 in TrkA residue num bering). Mutation of this site (Y-->F) eliminated PLC gamma activation (ind icating there are no other cryptic binding sites for PI,Cy in the DDR1 sequ ences) and markedly reduced the differentiative activity of the receptor. T his is in contrast to TrkA (or PDGFR beta/TrkA chimeras), where ablation of this pathway has no notable effect on PC12 cell morphogenic responses. Thu s, the activation of FRS2 and Shc (leading to MAPK activation) is weaker in the DDR1/TrkA chimeras than in TrkA alone, and the PLC gamma contribution becomes essential for full response. Nonetheless, both DDR1 JM regions cont ain potentially usable signaling sites, albeit they apparently are not acti vated directly in DDR1 (or DDR1 chimeras) in a ligand-dependent fashion. Th ese findings suggest that the DDR1 receptors do have signaling capacity but may require additional components or altered conditions to fully activate their kinase domains and/or sustain the activation of the JM sites.