Dual regulation of EDG1/S1P(1) receptor phosphorylation and internalization by protein kinase C and G-protein-coupled receptor kinase 2.
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.
Watterson
Kenneth R
KR
Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom.
Johnston
Evelyn
E
Chalmers
Claire
C
Pronin
Alexey
A
Cook
Simon J
SJ
Benovic
Jeffrey L
JL
Palmer
Timothy M
TM
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017931:DNA Primers; D005347:Fibroblasts; D049452:Green Fluorescent Proteins; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D007668:Kidney; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
D017931:DNA Primers; D017874:Immediate-Early Proteins; D008164:Luminescent Proteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010768:Phosphoserine; D019000:Phosphotyrosine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D051552:beta-Adrenergic Receptor Kinases; D013755:Tetradecanoylphorbol Acetate