The human vasoactive intestinal Peptide/Pituitary adenylate cyclase activating peptide receptor 1 (VPAC1): constitutive activation by mutations at threonine 343

The human vasoactive intestinal peptide/pituitary adenylate cyclase activating peptide receptor1 (VPAC1) belongs to the class II subfamily of G protein-coupled receptors. Specific changes by mutagenesis of a strictly conserved threonine (H) into lysine (K), proline (P) or alanine (A) at position 343 of the human VPAC1 receptor resulted in its constitutive activation with respect to cAMP production. Transfection of these mutants into Cos cells evoked a 3.5 fold-increase in the cAMP level as compared to cells transfected with the wild-type receptor. In contrast other mutants such as T343C, T343E or T343F were not constitutively activated. They were otherwise expressed at the cell surface of transfected nonpermeabilized cells. Double mutants were then constructed in which the T343K mutation was associated with a point mutation in the the N-terminal extracellular domain that totally abolished VIP binding or VIP-stimulated cAMP production i.e. E36A or D68A. The corresponding double mutants T343K-E36A and T343K-D68A were no longer constitutively activated. A control double mutant (T343K-D132A) with an unaltered dissociation constant for VIP and cAMP response to VIP, was still constitutively activated. Our findings demonstrate that constitutive activation of the VPAC1 receptor can be evoked by specific mutations of T343 at the junction of the second intracellular loop and fourth transmembrane segment. This constitutive activation appears to require the functional integrity of the N-terminal extracellular VIP binding domain.