Dual regulation of EDG1/S1P(1) receptor phosphorylation and internalization by protein kinase C and G-protein-coupled receptor kinase 2. |
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Authors: | Kenneth R Watterson Evelyn Johnston Claire Chalmers Alexey Pronin Simon J Cook Jeffrey L Benovic Timothy M Palmer |
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Affiliation: | Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom. |
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Abstract: | Here we demonstrate that phosphorylation of the sphingosine 1-phosphate (SSP) receptor "endothelial differentiation gene 1" (EDG1 or S1P(1)) receptor is increased in response to either SSP or phorbol 12-myristate 13-acetate (PMA) exposure but not lysophosphatidic acid. Phosphoamino acid analysis demonstrated that SSP stimulated the accumulation of phosphoserine and phosphothreonine but not phosphotyrosine. An inhibitor of PMA-stimulated EDG1 phosphorylation failed to block SSP-stimulated phosphorylation. Additionally, removal of 12 amino acids from the carboxyl terminus of EDG1 specifically reduced SSP- but not PMA-stimulated phosphorylation, suggesting that SSP and PMA increase EDG1 phosphorylation via distinct mechanisms. In vitro assays revealed that G-protein-coupled receptor kinase 2 may be at least partially responsible for SSP-stimulated EDG1 phosphorylation observed in intact cells. In addition, phosphorylation by PMA and SSP were associated with a loss of EDG1 from the cell surface by distinct mechanisms. Removal of 12 residues from the carboxyl terminus of EDG1 completely inhibited SSP-mediated internalization, suggesting that this domain dictates susceptibility to receptor internalization while retaining sensitivity to SSP-stimulated phosphorylation. Thus, we conclude that (a) EDG1 phosphorylation and internalization are controlled via independent mechanisms by agonist occupation of the receptor and protein kinase C activation, and (b) although determinants within the receptor's carboxyl-terminal tail conferring EDG1 sensitivity to agonist-mediated internalization and G-protein-coupled receptor kinase phosphorylation exhibit a degree of overlap, the two phenomena are separable. |
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