Two basic residues of the h-VPAC(1) receptor second transmembrane helix are essential for ligand binding and signal transduction

We mutated the vasoactive intestinal peptide (VIP) Asp(3) residue and two V PAC(1) receptor second transmembrane helix basic residues (Arg(188) and Lys (195)). VIP had a lower affinity for R188Q, R188L, K195Q, and R195I VPAC(1) , receptors than for VPAC, receptors, [Asn(3)] VIP and [Gln(3)] VIP had low er affinities than VIP for VPAC, receptors but higher affinities for the mu tant receptors; the two basic amino acids facilitated the introduction of t he negatively charged aspartate inside the transmembrane domain. The result ing interaction was necessary for receptor activation. 1/[Asn(3)] VIP and [ Gln(3)] VIP were partial agonists at VPAC, receptors; 21VIP did not fully a ctivate the K195Q, K195I, R188Q, and R188L VPAC, receptors; a VIP analogue ([Arg(16)] VIP) was more efficient than VIP at the four mutated receptors; and [Asn3] VIP and [Gln(3)] VIP were more efficient than VIP at the R188Q a nd R188L VPAC, receptors; the [Asp(3)] negative charge did not contribute t o the recognition of the VIP1 antagonist, [AcHis(1),D-Phe(2),Lys(15),Arg(16 ),Leu(27)] VIP (1-7)/growth hormone releasing factor (8-27). This is the fi rst demonstration that, to activate the VPAC, receptor, the Asp(3) side cha in of VIP must penetrate within the transmembrane domain, in close proximit y to two highly conserved basic amino acids from transmembrane 2.