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

Abstract

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.

Activation of Discoidin Domain Receptor 1 Facilitates the Maturation of Human Monocyte-Derived Dendritic Cells Through the TNF Receptor Associated Factor 6/TGF-β-Activated Protein Kinase 1 Binding Protein 1β/p38α Mitogen-Activated Protein Kinase Signaling Cascade

“…Forty-eight hours after re-stimulation, the cytotoxic activity of CD8 ϩ T cells was assessed in an 18-h 51 Cr-release assay. Ten million melanoma cells (target cells) in single-cell suspension were incubated with 100 Ci ϩ T cells (effector cells) generated by coculturing with mDCs at E:T ratios ranging from 4:1 to 80:1 in 96-well plates.…”

“…After an 18-h incubation, supernatants of each well were collected, and radioactivity was measured with a gamma counter (1480 WIZARTM 3"; PerkinElmer, Downer Grove, IL). Specific lysis was calculated by the formula: specific 51 Cr-release ϭ [(mean experimental cpm Ϫ mean spontaneous cpm)/(mean maximum cpm Ϫ mean spontaneous cpm)] ϫ 100%, in which spontaneous release represents cpm in supernatants from wells containing target cells with medium only, and maximum release represents cpm in supernatants from wells containing target cells in medium with 2% Triton X-100. At the same time, the supernatants from the 18-h coculture of unlabeled melanoma cells with effector cells (1:40) were collected and subjected to IFN-␥ ELISA in the Lymphokine Testing Laboratory.…”

“…2, A Ci with PBS. cubated with th mDCs at E:T an 18-h incubation, activity was measured inElmer, Downer Grove, ula: specific 51 Cr-release ϭ neous cpm)/(mean maximum %, in which spontaneous release ells containing target cells with merepresents cpm in supernatants from edium with 2% Triton X-100. At the same 18-h coculture of unlabeled melanoma cells ere collected and subjected to IFN-␥ ELISA in Laboratory.…”

Mapping of Epitopes in Discoidin Domain Receptor 1 Critical for Collagen Binding

“…The 37-amino acid insert in DDR1b shows the motif LLXNPXY, which can interact with the phosphotyrosine-binding domain of the Shc adapter protein upon collagen-induced tyrosine phosphorylation (1). Binding of the protein FRS2 has been shown to a chimeric molecule containing the juxtamembrane region of DDR1a (22). Furthermore, recent data imply that the Wnt-5a pathway may overlap with DDR1 signaling (23).…”

Discoidin Domain Receptor 1

“…A sixth isoform that lacks part of the extracellular domain has been described in rat testis. 15 These isoforms exhibit differences in the extent of glycosylation, 16 phosphorylation, 6,17 protein interactions, 18,19 and expression patterns and functions. 17 DDR1 regulates adhesion, 17,20 migration, 17,[21][22][23][24] proliferation and apoptosis, 24,25 cell morphogenesis and differentiation, 21,24 and ECM remodeling.…”