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 motif in the extracellular domain, an unusually long cytoplasmic juxtamembrane (JM) region, and a kinase domain that is 45% identical to that of the NGF receptor, 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 the shorter receptor. One class of ligands for the DDR receptors has recently been identified as being derived from the collagen family, but neither native 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 hPDGFRbeta fused to the transmembrane and intracellular regions of DDR1, also fails to mediate neuronal-like differentiation in stably transfected PC12 cells and is only weakly autophosphorylated. However, chimeric receptors, which are composed of combinations of intracellular regions from DDR1 and TrkA (with the extracellular domain of hPDGFRbeta), in some cases provided ligand (PDGF) -inducible receptor responses. Those with the TrkA kinase domain and the DDR1 JM regions were able to produce differentiation to varying degrees, whereas the opposite combination did not. Analysis of the signaling responses of the two chimeras with DDR1 JM sequences (with and without the insert) indicated that the shorter sequence bound and activated FRS2 whereas the insert-containing form activated Shc instead. Both activated PLCgamma through the carboxyl-terminal tyrosine of the TrkA domain (Y785 in TrkA residue numbering). Mutation of this site (Y-->F) eliminated PLCgamma activation (indicating there are no other cryptic binding sites for PLCgamma in the DDR1 sequences) and markedly reduced the differentiative activity of the receptor. This is in contrast to TrkA (or PDGFRbeta/TrkA chimeras), where ablation of this pathway has no notable effect on PC12 cell morphogenic responses. Thus, the activation of FRS2 and Shc (leading to MAPK activation) is weaker in the DDR1/TrkA chimeras than in TrkA alone, and the PLCgamma contribution becomes essential for full response. Nonetheless, both DDR1 JM regions contain potentially usable signaling sites, albeit they apparently are not activated directly in DDR1 (or DDR1 chimeras) in a ligand-dependent fashion. These 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.
Foehr
E D
ED
Departments of Physiology and Biophysics, College of Medicine, University of California, Irvine, California 92697-4560, USA.
Tatavos
A
A
Tanabe
E
E
Raffioni
S
S
Goetz
S
S
Dimarco
E
E
De Luca
M
M
Bradshaw
R A
RA
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D000070818:Discoidin Domain Receptors; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism; D016716:PC12 Cells; D051966:Phospholipase C gamma; D010766:Phosphorylation; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases; D020917:Receptor, trkA / Q000235:genetics / Q000378:metabolism*; D011975:Receptors, Mitogen / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000378:metabolism
D007527:Isoenzymes; D011975:Receptors, Mitogen; D011993:Recombinant Fusion Proteins; D000070818:Discoidin Domain Receptors; D020794:Receptor Protein-Tyrosine Kinases; D020917:Receptor, trkA; D017479:Receptors, Platelet-Derived Growth Factor; D010738:Type C Phospholipases; D051966:Phospholipase C gamma