Functional characterisation of the human S1P₄G protein-coupled receptor

The human G protein-coupled receptor (GPCR) S1P4 (formerly named Edg6) was identified as an orphan receptor and assigned to the Edg family on the basis of sequence similarity with other Edg receptors. S1P4 was recently shown to couple to Gαi and Gα12/13 G proteins in response to stimulation by the lysophospholipid, sphingosine-1-phosphate (S1P). This thesis describes functional characterisation of the S1P4 receptor using a modified [35S]GTPγS binding assay. CHO-K1 cells were stably transfected to express S1P4, or a fusion protein between S1P4 and a pertussis toxin-resistant form of Gαi1. S1P4 exhibited significant constitutive activity and treatment with SIP or phytoSIP further stimulated the receptor in a dose-dependent manner. Pertussis toxin treatment demonstrated that both S1P4 constitutive activity and the effects of SIP were transduced via endogenous Gαi G proteins, whilst the SlP4-Gαi1 fusion signalled via the tethered, pertussis toxin-insensitive G protein. Residue was shown to be important in governing S1P4 ligand selectivity; mutation of the naturally occurring glutamic acid to glutamine attenuated the ability of the receptor to respond to S1P and conferred sensitivity to the related compound, lysophosphatidic acid. Since most cell types express endogenous S1P and LPA family receptors, the profile of Edg family receptor expression in CHO-K1 cells was investigated. Transcripts for S1P1,2,4 and LPA1 receptors were detected using RT-PCR with species-specific oligonucleotides. Further investigations into S1P4 constitutive activity, which were undertaken using an inducible expression system, demonstrated direct correlation between S1P4 expression and constitutive activity. This suggested that S1P4 constitutive activity represents an inherent property of this receptor. In common with other naturally constitutively active GPCRs, S1P4 lacks a generally conserved cysteine residue within the first extracellular loop. It is proposed that this may account for S1P4 constitutive activity and may be relevant to the in vivo function of this receptor