Probing the function of residue 3.60 in peptide ligand-GPCRs

Richard Logan, Rachel Kendrick, Mark Wheatley. University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK

With the increasing availability of GPCR crystallographic data, identification of structural elements within loop regions is now possible. Secondary structure is present in both extracellular loops (reviewed in Wheatley et al., 2012) and intracellular loop regions of GPCRs. Intracellular loop 2 (ICL2) has been revealed to contain a short helical segment in many GPCRs. This helix appears to be a dynamic feature, given that distinct ICL2 conformations are observed for the dopamine D3 receptor in each of the two receptor molecules of the crystallographic asymmetric unit (Chien et al., 2010). It is noteworthy that in monoamine ligand-GPCRs, residue 3.60 located centrally within ICL2 is almost exclusively tyrosine. In contrast, tyrosine is excluded at this position in peptide ligand-GPCRs. The role of this residue was investigated in this study.

A QuikChange™ (Stratagene) site-directed mutagenesis strategy was used to generate point mutations at position 3.60 in N-terminal HA-tagged peptide-ligand GPCRs within mammalian expression vector pcDNA3.1(+) (Invitrogen). The human ghrelin receptor (ghrelin-R) and human vasopressin 1a receptor (V1aR) are both Gq/11 coupled peptide-ligand GPCRs with substantial constitutive activity (CA) (50-60% of Emax), and little/no constitutive activity, respectively. The contribution of the side chain properties at position 3.60 to receptor structure and function were assessed by competition binding assay (V1aR constructs only) and inositol phosphates accumulation assay of transiently transfected HEK293Tcells. Cell-surface expression was determined by ELISA. All data are expressed as mean ± s.e.m. of experiments performed in triplicate.

V1aR receptor constructs T3.60A and T3.60S represent commonly observed residues at position 3.60 in peptide ligand-GPCRs and demonstrated wildtype-like function. T3.60F was also well tolerated while the substitution T3.60Y, typical of monoamine ligand-GPCRs, demonstrated increased cell-surface expression compared to wildtype (152 ± 3%). Ghrelin-R constructs A3.60F and A3.60Y possessed sever decreases in CA of 87 ± 14% and 71 ± 18% respectively, but exhibited wildtype cell-surface expression. Constructs were dose-responsive to ghrelin challenge but maximum signalling was impaired in A3.60Y (63 ± 11% of wildtype Emax) whereas A3.60F was wildtype-like.

In conclusion, introduction of Tyr3.60, typical of monoamine ligand-GPCRs, apparently stabilised the V1aR structure leading to increased cell-surface expression. In the ghrelin-R, an aromatic ring at position 3.60 (Phe) reduced constitutive signalling, whereas the additional presence of an hydroxyl (Tyr) also reduced maximal signalling.

Chien EY, Liu W, Zhao Q, Katrich V, Han GW, Hanson MA et al. (2010). Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist. Science 330: 1091–1095

Wheatley M, Wootten D, Conner MT , Simms J, Kendrick R, Logan R et al. (2012) Lifting the lid on GPCRs: the role of extracellular loops. Br J Pharmacol 165: 1688-1703