Agonist-dependent consequences of proline to alanine substitution in the transmembrane helices of the calcitonin receptor

Abstract

Background and purpose: Transmembrane proline (P) residues in family A G protein-coupled receptors (GPCRs) form functionally important kinks in their helices. These residues are little studied in family B GPCRs but experiments with the VPAC1 receptor and calcitonin receptor-like receptor (CL) show parallels with family A receptors. We sought to determine the function of these residues in the insert negative form of the human calcitonin receptor, a close relative of CL.

Experimental approach: Proline residues within the transmembrane domains of the calcitonin receptor (P246, P249, P280, P326, P336) were individually mutated to alanine (A) using site-directed mutagenesis. Receptors were transiently transfected into Cos-7 cells using polyethylenimine and salmon and human calcitonin-induced cAMP responses measured. Salmon and human calcitonin competition binding experiments were also performed and receptor cell-surface expression assessed by whole cell ELISA.

Key results: P246A, P249A and P280A were wild-type in terms of human calcitonin-induced cAMP activation. P326A and P336A had reduced function (165 and 12-fold, respectively). In membranes, human calcitonin binding was not detectable for any mutant receptor but in whole cells, binding was detected for all mutants apart from P326A. Salmon calcitonin activated mutant and wild-type receptors equally, although B(max) values were reduced for all mutants apart from P326A.

Conclusions and implications: P326 and P336 are important for the function of human calcitonin receptors and are likely to be involved in generating receptor conformations appropriate for agonist binding and receptor activation. However, agonist-specific effects were observed , implying distinct conformations of the human calcitonin receptor.

Figure 1

Alignment of the insert negative form of the human calcitonin receptor (CT_(a)) with its closest relative, the CL showing the conserved transmembrane proline residues in white text on black. Alignment was performed by ClustalW. Predicted signal sequences are italicized, transmembrane (TM) regions are underlined.

Figure 2

Stimulation of cAMP production by human calcitonin at calcitonin receptor proline mutants or wild-type (WT) calcitonin receptors, as indicated (a–e) expressed as a percentage of the cAMP response generated by 50 μM forskolin. Data are mean±s.e.m. of four to nine experiments, performed in duplicate or triplicate.

Figure 3

Binding of ¹²⁵I-hCT (a) or ¹²⁵I-sCT (b, c, d) to membranes or ¹²⁵I-hCT (e, f) binding to cells transiently transfected with wild-type (WT) or mutant calcitonin receptors. Experiments were repeated three to nine times. Data shown are representative and data points are mean±s.e.m. of duplicate or triplicate points.

Figure 4

Cell-surface expression of HA-tagged wild-type (WT) or mutant calcitonin receptors. Mutant expression was normalized to WT expression and combined data are shown. Experiments were performed in quadruplicate and data shown are the mean±s.e.m. of three to four separate experiments. ^*P<0.05, ^**P<0.01 compared to WT by non-parametric one-way ANOVA (Kruskal–Wallis test) followed by Dunn's multiple comparison test.

Figure 5

Effect of DNA dilution on human calcitonin-stimulated cAMP production at wild-type (WT) (a) or P246A (b) calcitonin receptors. The legends show the amount of receptor DNA transfected into each well. Experiments were repeated three times. Data shown are representative and data points are mean±s.e.m. of duplicate points.