Differential determinants for coupling of distinct G proteins with the class B secretin receptor

Abstract

The secretin receptor is a prototypic class B G protein-coupled receptor that is activated by binding of its natural peptide ligand. The signaling effects of this receptor are mediated by coupling with Gs, which activates cAMP production, and Gq, which activates intracellular calcium mobilization. We have explored the molecular basis for the coupling of each of these G proteins to this receptor using systematic site-directed mutagenesis of key residues within each of the intracellular loop regions, and studying ligand binding and secretin-stimulated cAMP and calcium responses. Mutation of a conserved histidine in the first intracellular loop (H157A and H157R) markedly reduced cell surface expression, resulting in marked reduction in cAMP and elimination of measurable calcium responses. Mutation of an arginine (R153A) in the first intracellular loop reduced calcium, but not cAMP responses. Mutation of a dibasic motif in the second intracellular loop (R231A/K232A) had no significant effects on any measured responses. Mutations in the third intracellular loop involving adjacent lysine and leucine residues (K302A/L303A) or two arginine residues separated by a leucine and an alanine (R318A/R321A) significantly reduced cAMP responses, while the latter also reduced calcium responses. Additive effects were elicited by combining the effective mutations, while combining all the effective mutations resulted in a construct that continued to bind secretin normally, but that elicited no significant cAMP or calcium responses. These data suggest that, while some receptor determinants are clearly shared, there are also distinct determinants for coupling with each of these G proteins.

Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies

“…The KxxK 6.35 motif may interact with the C-terminus of Gαs, aided by flexing of TM6 around P 6.42 (class B) or P 6.50 (class A). This interaction is born out by MD simulations, but the analogous residues in the β 2 -AR–Gs complex (PDB code 3SN6) are poorly resolved and so the interpretation should be used with care, despite the observations from mutagenesis experiments that implicate K 6.32 and K 6.35 in G-protein coupling in both class A [36,46–48] and class B [39,49–52] GPCRs. For this reason, it is important to note that the alignment is based on mutagenesis results throughout the transmembrane region and not in just one region, such as the G-protein coupling region.…”

Mapping spatial approximations between the amino terminus of secretin and each of the extracellular loops of its receptor using cysteine trapping

“…Cell surface expression of secretin receptor mutants that exhibited no saturable binding. COS‐1 cells transiently expressing each of the noted secretin receptor mutant constructs were analyzed by flow cytometry using an amino‐terminal region secretin receptor antibody (18). Data represent percentage expression levels of each mutant relative to wild type receptor, expressed as means ± se of 3 independent experiments.…”

MOLECULAR EVOLUTION OF GPCRS: Secretin/secretin receptors

“…However, mutation analysis showed that this motif did not affect the calcium responses in human SCTR (Garcia et al 2012). Although Lys242 is conserved in secretin receptors, Arg241 is not conserved across different species, suggesting that this motif is not involved in controlling G-protein functioning in SCTR.…”

“…It has been reported that the His165 in TM2 is essential for the surface expression of secretin receptor. Mutation of this His residue (H166A or H166R) in human SCTR decreases the ligand-binding affinity, as well as cAMP response and calcium signaling, thus suggesting the poor surface expression of these mutants (Garcia et al 2012). Lys312 and Leu313 residues in the ICL3 are important for cAMP signaling in human SCTR (Garcia et al 2012) and also in other secretin GPCRs (Mathi et al 1997, Marie et al 2003.…”

“…6, the xCxR motif is well conserved from fish to mammalian secretin receptors. Reported to be important for G-protein functioning (Garcia et al 2012), mutation of the Arg162 residue in this motif reduces the cAMP responses without abolishing the ligand-binding ability in the rat calcitonin receptor-like receptor (Conner et al 2006) and the rat glucagon receptor (Cypess et al 1999). However, this mutation in human SCTR did not impair cAMP signaling, but caused a complete loss of calcium responses (Garcia et al 2012).…”