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 activat ing peptide receptor1 (VPAC1) belongs to the class II subfamily of G; prote in-coupled receptors. Specific changes by mutagenesis of a strictly conserv ed threonine (H) into lysine (K), proline (P) or alanine (A) at position 34 3 of the human VPAC1 receptor resulted in its constitutive activation with respect to cAMP production. Transfection of these mutants into Cos cells ev oked 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, T343 E 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 poi nt mutation in the the N-terminal extracellular domain that totally abolish ed VIP binding or VIP-stimulated cAMP production i.e. E36A or D68A The corr esponding double mutants T343K-E36A and T343H-D68A were no longer constitut ively activated. A control double mutant (T343K-D132A) with an unaltered di ssociation constant for VIP and cAMP response to VIP, was still constitutiv ely 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 co nstitutive activation appears to require the functional integrity of the N- terminal extracellular VIP binding domain. (C) 1999 Academic Press.