Photoaffinity Labeling Demonstrates Physical Contact between Vasoactive Intestinal Peptide and the N-terminal Ectodomain of the Human VPAC1 Receptor

Abstract

Vasoactive intestinal peptide (VIP)1 is a prominent neuropeptide present in the central and peripheral nervous systems as well as in immune cells (1-3). In consonance with its ubiquitous distribution, VIP controls a large array of biological functions (1-3). A non-exhaustive list includes exocrine secretions, release of hormones, neuronal excitation, relaxation of muscles, metabolism, immune function, inflammatory response, growth control of fetuses, embryonic brain development, and tumor cell biology (1-5). VIP action on its numerous target cells is mediated by two serpentine G protein-coupled receptors referred to as VPAC1 and VPAC2 (6). These receptors also have high affinity for pituitary adenylate cyclase-activating polypeptide, another neuropeptide structurally related to VIP. VPAC receptors belong to the class II subfamily within the superfamily of G protein-coupled receptors (6 -8). It includes receptors for peptides structurally related to VIP (glucagon, glucagons-like peptides, secretin, gastric inhibitory polypeptide, pituitary adenylate cyclase-activating polypeptide, growth hormone-releasing factor), receptors for other peptides such as parathyroid hormone, calcitonin or corticotropin-releasing factor (7), and the so-called epidermal growth factor-TM7 or LNB-TM7 (9) receptors bearing unusually large and complex N-terminal extracellular domains.The VPAC1 receptor is a prototypical class II peptide receptor that has been extensively studied by site-directed mutagenesis and molecular chimerism with respect to characterization of VIP binding domains (for review see Ref. 6), the existence of selectivity filters for low affinity agonists (10, 11) and identification of cytoplasmic domains required for activation of adenylyl cyclase (12). These studies showed that the N-terminal extracellular domain of hVPAC1 receptor is necessary although not sufficient for high affinity VIP binding (6). Moreover, microdomains consisting of small clusters of amino acids located in the N-terminal ectodomain (11) or at the junction of extra loop 1 and transmembrane helix 3 (10) were characterized as selectivity filters restricting access of VIP-related peptides to the receptor. A three-dimensional model of a large part of the N-terminal ectodomain of hVPAC1 receptor has been recently developed (13). Despite these extensive studies of the structure-function relationship of hVPAC1 receptor, we do not yet know the physical sites of interaction between VIP and its receptor.In this context, we developed a photoaffinity probe of VIP by substituting para-benzoyl-L-Phe (Bpa) for tyrosine 22. This site of incorporation of the benzophenone group, a photoactivable group of choice for high efficiency covalent labeling of proteins (14), was selected for several reasons. (i) Tyr 22 is present in the central part of the 28-amino acid VIP, a domain that has been shown previously by alanine scanning to contain several amino acids that are crucial for VIP binding (15). (ii) Alanine scanning showed that Tyr 22 itself is not important for the b...

Diffuse Pharmacophoric Domains of Vasoactive Intestinal Peptide (VIP) and Further Insights into the Interaction of VIP with the N-terminal Ectodomain of Human VPAC1 Receptor by Photoaffinity Labeling with [Bpa6]-VIP

“…Our data (present paper and Ref. 8) are consistent with a model in which the central part of VIP, at least between Phe 6 and Tyr 22 , lies in the binding groove identified in the N-terminal ectodomain of the hVPAC1 receptor (Fig. 6).…”

“…Together with a previous photoaffinity labeling study (8), these data indicate that the central part of VIP lies in a binding groove within the N-terminal ectodomain of the hVPAC1 receptor. Additional studies with new photoaffinity probes of VIP are needed to further delineate the sites of interaction between the central part of VIP and this binding groove for which a structural model has been already developed (3,7) and also to identify new anchor points of the N-terminal domain of VIP in the receptor core.…”

“…6). Another most meaningful aspect of the present study is related to the fact that the contact segment (8). This is illustrated in Fig.…”

“…The hypothesis that the VIP-binding cleft could reside between the hVPAC1 receptor N-terminal ectodomain for the central part of VIP (this paper and Ref. 8) and the receptor body for the N-terminal domain of VIP is reminiscent of the situation described for some other class II peptide receptors (22)(23)(24)(25).…”

“…A three-dimensional model of the N-terminal ectodomain of the hVPAC1 receptor has been developed suggesting the existence of a VIP-binding groove within this domain (7). Despite these extensive studies of the structure-function relationship of hVPAC1 receptor, the physical sites of interaction between VIP and its receptors had remained elusive until recent photoaffinity showing labeling experiments that the side chain of position 22 of VIP is in direct contact with one edge of the putative binding groove in the N-terminal ectodomain (8).…”