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Functional analysis of discoidin domain receptor 1: effect of adhesion on DDR1 phosphorylation
Corine G M L'hôte1, Peter H Thomas, Trivadi S Ganesan
1ICRF Molecular Oncology Laboratories, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK.
Abstract:
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.
Insights
Cellular context, specifically matrix interactions, controls the slow collagen-induced phosphorylation of discoidin domain receptor 1 (DDR1). Detached cells show rapid DDR1 phosphorylation, unlike adherent cells, suggesting matrix adhesion inhibits this receptor tyrosine kinase.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase (RTK) primarily activated by soluble fibrillar collagen.
- DDR1 phosphorylation kinetics are unusually slow, with peak activity at 90 minutes.
Purpose of the Study:
- Investigate the reasons behind the slow phosphorylation kinetics of DDR1.
- Determine the cellular factors influencing DDR1 activation by collagen.
Main Methods:
- Examined DDR1 phosphorylation in various cell lines (T47D, HCT116, COLO201, K562) under adherent and detached conditions.
- Utilized pervanadate treatment to assess phosphatase activity.
- Investigated downstream signaling pathways (MAPK) and used chimeric receptors (TrkA-DDR1).
Main Results:
- Endogenous DDR1 exhibits slow collagen-induced phosphorylation in adherent cells but rapid phosphorylation in detached cells.
- Cellular context, particularly matrix interaction (e.g., fibronectin), dictates phosphorylation kinetics.
- Phosphatase activity likely inhibits or delays DDR1 phosphorylation; downstream signals do not solely rely on the MAPK pathway.
Conclusions:
- DDR1 phosphorylation kinetics are dependent on the cellular context, a novel finding for RTKs.
- Cell-matrix interactions, not cell-cell contacts, are implicated in the delayed phosphorylation of DDR1.
- This study reveals a unique regulatory mechanism for DDR1 activation by collagen.