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 S1P3 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) S1P3 could be phosphorylated in vitro by G-protein-coupled receptor kinase 2 (GRK2), a role of S1P3 phosphorylation in controlling S1P3-Gq/11 coupling was excluded since A) a phosphorylation-resistant S1P3 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 S1P3 phosphorylation. S1P3 phosphorylation was also not required for receptor sequestration away from the cell surface. Together, these data suggest that S1P3 function is not subject to conventional regulation by GRK phosphorylation and that novel aspects of S1P3 function distinct from classical G-protein coupling and receptor internalisation may be controlled its carboxyl-terminal domain.