Phosphorylation-independent internalisation and desensitisation of the human sphingosine-1-phosphate receptor S1P3.
Here we demonstrate that phosphorylation of the sphingosine-1-phosphate (S1P) receptor S1P(3) is increased specifically in response to S1P. Truncation of the receptor's carboxyl-terminal domain revealed that the presence of a serine-rich stretch of residues between Leu332 and Val352 was essential to observe this effect. Although agonist-occupied wild-type (WT) S1P(3) could be phosphorylated in vitro by G-protein-coupled receptor kinase 2 (GRK2), a role of S1P(3) phosphorylation in controlling S1P(3)-G(q/11) coupling was excluded since A) a phosphorylation-resistant S1P(3) mutant desensitised in a manner indistinguishable from the WT receptor and was phosphorylated to a greater extent than the WT receptor by GRK2 in vitro, and B) co-expression with GRK2 or GRK3 failed to potentiate S1P(3) phosphorylation. S1P(3) phosphorylation was also not required for receptor sequestration away from the cell surface. Together, these data suggest that S1P(3) function is not subject to conventional regulation by GRK phosphorylation and that novel aspects of S1P(3) function distinct from classical G-protein coupling and receptor internalisation may be controlled its carboxyl-terminal domain.
Rutherford
Claire
C
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland, UK.
Ord-Shrimpton
Fiona U
FU
Sands
William A
WA
Pediani
John D
JD
Benovic
Jeffrey L
JL
McGrath
John C
JC
Palmer
Timothy M
TM
D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D000939:Epitopes / Q000737:chemistry; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D049349:Receptors, Lysosphingolipid / Q000737:chemistry / Q000502:physiology*; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D000939:Epitopes; D016328:NF-kappa B; D049349:Receptors, Lysosphingolipid; D012694:Serine; D002118:Calcium