The 5-HT7 receptor is a G-protein coupled receptor that has multiple biological roles in mammalian systems, including the setting of circadian rhythms (Barnes & Sharp, 1999). There are four known mammalian splice variants (5-HT_7(a-d)), differing only in their carboxy-termini, which are predicted to be intracellular (Heidmann et al, 1997), each containing a different number of potential phosphorylation sites, suggesting this domain may have a significant role in receptor modulation by kinases.

This study aimed to identify the specific amino acid residues of the large intracellular loop and C-terminal domain of the h5-HT_(7b) receptor that are phosphorylated by PKA, PKC, PKG and CaMKII using phosphoamino acid analysis (PAA; Kamps et al., 1989) and site-directed mutagenesis.

cDNA sequences encoding the third cytoplasmic loop (loop: Q261-T328) and C-terminal (cterm: N388-STOP) domains of h5-HT_7(b) were expressed as GST fusion proteins, purified/immobilised from lysed cultures using glutathione agarose beads, and subsequently labelled with [³²P]-ATP, using a standard in vitro kinase assay (for each assay n3; MacDonald et al., 1997). Labelled samples were resolved by SDS-PAGE, transferred onto PVDF and the [³²P]-labelled bands excised and acid hydrolysed (5.7M HCl, 1hr, 110^oC) before resolution on 2D cellulose thin layer chromatography (TLC) plates (1^st dimension pH1.9, 20 min, 1.5 kV; 2^nd dimension pH 3.5, 13 min, 1.6 kV). Autoradiography of TLC plates revealed the identity of residue types phosphorylated in each reaction by comparison of the migration patterns of our [³²P]-labelled samples with those of stained 'cold' phospho-amino acids.

PAA revealed serine residues to be the phosphorylation target of both PKA and PKG in the cterm and of PKC in the loop. In addition, PKC phosphorylation of the cterm appeared to occur at both serine and threonine residues. To date, no PAA data for CaMKII phosphorylation has been resolved.

QuikChange site-directed mutagenesis (Stratagene) was employed to mutate potential kinase target residues to alanine, and the in vitro kinase assay repeated. S311A was found to completely remove phosphorylation of cterm by PKA and PKG and reduced phosphorylation compared to wild-type (WT) cterm by CaMKII, suggesting that multiple stimuli may elicit the same cellular response via this receptor. S397A and T395A were both found to reduce PKC phosphorylation of the cterm individually. Phosphorylation of the loop by PKC was reduced (compared to WT) in the S317A construct, but no other single serine or threonine mutation gave a reduction in phosphorylation levels.

Future work includes the identification of the remaining kinase substrates in these sequences, followed by in vivo phosphorylation of full-length cloned h5-HT_(7b) in transiently transfected cell lines.

Barnes, NM & Sharp, T (1999) Neuropharm., 38:1083-1152.
Heidmann, D.E, et al. (1997) J Neurochem., 68:1372-1381.
Kamps M.P. & Sefton B.M. (1989) Anal Biochem., 176:22-27.
McDonald B.J. & Moss, S.J. (1997) Neuropharm., 36:1377-1385.