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

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 Bmax 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-specific effects were observed implying distinct conformat human calcitonin receptor. effects were observed, implying distinct conformations of the human calcitonin receptor.