Functional analysis of discoidin domain receptor 1: effect of adhesion on DDR1 phosphorylation.
Discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase (RTK), has been shown to be activated mainly by soluble fibrillar collagen. Unusually, the kinetics of phosphorylation of the receptor is slow, with maximal phosphorylation observed after 90 min. To understand the reasons for slow phosphorylation of the receptor, we examined several cell lines under different conditions. We confirm that endogenous DDR1 is phosphorylated slowly by collagen in adherent T47D and HCT116 cells. In detached and resuspended cells, collagen induced rapid phosphorylation of DDR1. This was further confirmed with a semiadherent cell line (COLO201) and one that grows as a suspension (K562), both of which express endogenous DDR1. Replating K562 on fibronectin to mimic adherent conditions altered the kinetics of phosphorylation from rapid to slow, similar to those of adherent cells. The slow kinetics of phosphorylation in the adherent state was probably not due to cell-cell contacts because EDTA had no major effect. However, pervanadate in the absence of collagen was able to induce strong DDR1 phosphorylation, indicating that a phosphatase may inhibit or delay the phosphorylation of DDR1. Further, downstream signals after phosphorylation of DDR1 by collagen were not transmitted through the classical mitogen-activated protein kinase pathway. In addition, a chimeric TrkA-DDR1 receptor failed to become phosphorylated on stimulation with nerve growth factor (NGF), although it dimerized normally. This is the first RTK whose kinetics of phosphorylation is dependent on cellular context. The interaction of the cells with the matrix, rather than cell-cell contact, is probably responsible for the inhibition of phosphorylation.
L'hôte
Corine G M
CG
ICRF Molecular Oncology Laboratories, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK.
Thomas
Peter H
PH
Ganesan
Trivadi S
TS
D016475:3T3 Cells; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D003094:Collagen / Q000378:metabolism; D000070818:Discoidin Domain Receptors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020014:K562 Cells; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases; D011975:Receptors, Mitogen / Q000378:metabolism*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology
D004791:Enzyme Inhibitors; D011975:Receptors, Mitogen; C076715:pervanadate; D014638:Vanadates; D003094:Collagen; D019869:Phosphatidylinositol 3-Kinases; D000070818:Discoidin Domain Receptors; D020794:Receptor Protein-Tyrosine Kinases; D017027:Protein Tyrosine Phosphatases