Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) activate two shared receptors, VPAC1 and VPAC2. Activation of VPAC1 has been implicated in elevating glucose output, whereas activation of VPAC2 may be involved in insulin secretion. A hypothesis that a VPAC2-selective agonist would enhance glucose disposal by stimulating insulin secretion without causing increased hepatic glucose production was tested using a novel selective agonist of VPAC2. This agonist, BAY 55-9837, was generated through site-directed mutagenesis based on sequence alignments of PACAP, VIP, and related analogs. The peptide bound to VPAC2 with a dissociation constant (K d ) of 0.65 nmol/l and displayed >100-fold selectivity over VPAC1. BAY 55-9837 stimulated glucose-dependent insulin secretion in isolated rat and human pancreatic islets, increased insulin synthesis in purified rat islets, and caused a dose-dependent increase in plasma insulin levels in fasted rats, with a half-maximal stimulatory concentration of 3 pmol/kg. Continuous intravenous or subcutaneous infusion of the peptide reduced the glucose area under the curve following an intraperitoneal glucose tolerance test. The peptide had effects on intestinal water retention and mean arterial blood pressure in rats, but only at much higher doses. BAY 55-9837 may be a useful therapy for the treatment of type 2 diabetes.

GnRH plays a pivotal role in the reproductive system, and GnRH analogs have wide therapeutic applications ranging from the treatment of prostatic cancer to infertility. Determination of the predicted structure of the GnRH receptor (GnRHR) would illuminate the mechanisms of receptor activation and regulation and allow directed design of improved GnRH analogs. We report the cloning of a cDNA representing the mouse GnRHR and confirm its identity using Xenopus oocyte expression. Injection of sense RNA transcript leads to the expression of a functional, high affinity GnRHR. Expression of the GnRHR using gonadotrope cell line RNA, however, is blocked by an antisense oligonucleotide. In situ hybridization in the rat anterior pituitary reveals a characteristic GnRHR distribution. The nucleotide sequence encodes a 327-amino acid protein which has the seven putative transmembrane domains characteristic of G protein-coupled receptors, but which lacks a typical intracellular C-terminus. The unusual structure and novel potential regulatory domain of the GnRHR may explain unique aspects of its signal transduction and regulation.

GnRH plays a pivotal role in the reproductive system, and GnRH analogs have wide therapeutic applications ranging from the treatment of prostatic cancer to infertility. Determination of the predicted structure of the GnRH receptor (GnRHR) would illuminate the mechanisms of receptor activation and regulation and allow directed design of improved GnRH analogs. We report the cloning of a cDNA representing the mouse GnRHR and confirm its identity using Xenopus oocyte expression. Injection of sense RNA transcript leads to the expression of a functional, high affinity GnRHR. Expression of the GnRHR using gonadotrope cell line RNA, however, is blocked by an antisense oligonucleotide. In situ hybridization in the rat anterior pituitary reveals a characteristic GnRHR distribution. The nucleotide sequence encodes a 327-amino acid protein which has the seven putative transmembrane domains characteristic of G protein-coupled receptors, but which lacks a typical intracellular C-terminus. The unusual structure and novel potential regulatory domain of the GnRHR may explain unique aspects of its signal transduction and regulation.

Pituitaryadenylate cyclase-activating peptide (PACAP) has a specific receptor PAC1 and shares two receptors VPAC1 and VPAC2 with vasoactive intestinal peptide (VIP). VPAC2 activation enhances glucose-induced insulin release while VPAC1 activation elevates glucose output. To generate a large pool of VPAC2 selective agonists for the treatment of type 2 diabetes, structure-activity relationship studies were performed on PACAP, VIP, and a VPAC2 selective VIP analog. Chemical modifications on this analog that prevent recombinant expression were sequentially removed to show that a recombinant peptide would retain VPAC2 selectivity. An efficient recombinant expression system was then developed to produce and screen hundreds of mutant peptides. The 11 mutations found on the VIP analog were systematically replaced with VIP or PACAP sequences. Three of these mutations, V19A, L27K, and N28K, were sufficient to provide most of the VPAC2 selectivity. C-terminal extension with the KRY sequence from PACAP38 led to potent VPAC2 agonists with improved selectivity (100 -1000-fold). Saturation mutagenesis at positions 19, 27, 29, and 30 of VIP and chargescanning mutagenesis of PACAP27 generated additional VPAC2 selective agonists. We have generated the first set of recombinant VPAC2 selective agonists described, which exhibit activity profiles that suggest therapeutic utility in the treatment of diabetes.Pituitary adenylate cyclase-activating polypeptide (PACAP), 1 originally isolated from ovine hypothalamus (1) by following pituitary adenylate cyclase activation, belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) family of peptides (2). These peptides are expressed as part of larger proteins that are processed by proteolysis followed by Cterminal amidation to generate the mature amidated peptides (Fig. 1). PACAP exists as a 38-residue form (PACAP38), and as a shorter form corresponding to the N-terminal 27 amino acids of PACAP38 (PACAP27). Both forms of PACAP bind to and activate the G-protein-coupled receptors PAC1, VPAC1, and VPAC2, whereas the related 28-mer peptide VIP only recognizes VPAC1 and VPAC2 (3). Activation of multiple receptors by PACAP or VIP has broad physiological effects on nervous, endocrine, cardiovascular, reproductive, muscular, and immune systems (4). Thus, clinical applications will require selective activation of a particular receptor to minimize potential side effects mediated by the other receptors. For example, we have previously demonstrated that VPAC2 activation induces glucose-dependent insulin secretion without the undesired side effects mediated by VPAC1 such as watery diarrhea (5). Therefore, to provide a potential therapy for type 2 diabetes, VPAC2 selectivity is an absolute requirement.We sought to identify key determinants of VPAC2 selectivity and generate a peptide with the minimum number of mutations. However, structure-function relationship studies on VIP and PACAP (6 -15) have been restricted by the number of peptides that can be tested due to limitations of peptide synth...

Sertraline is a new, selective serotonin (5HT) uptake inhibitor with antidepressant activity. The effect of chronic administration of sertraline on 5HT-2 receptors in rat cortex was compared with that of the tricyclic antidepressant, amitriptyline. 5HT-2 receptors were evaluated in binding assays using [3H]-ketanserin and in functional assays of transmembrane signaling, hydrolysis of phosphoinositides. The daily injection of 17 mg/kg sertraline induced a desensitization of 5HT-2-mediated phosphoinositide hydrolysis after 28, but not 21, days. The administration of 1.2 mg/kg/day via continuous release pumps caused a more rapid desensitization. Amitriptyline administered chronically also produced a desensitization of the 5HT-2-mediated phosphoinositide hydrolysis response. A decrease in the density of 5HT-2 binding sites accompanies the functional desensitization after amitriptyline, but changes in 5HT-2 binding sites were not detected after chronic sertraline administration. Studies of the mechanism of action of sertraline show that the desensitization of the phosphoinositide hydrolysis response is homologous in nature, and that it is not secondary to changes in the synthesis of precursor lipids. Other possibilities such as alterations in coupling efficiency or in the activity of effector enzymes are currently being considered. The present results suggest a new postsynaptic action of antidepressant drugs at central 5HT-2 receptors (i.e., changes in 5HT-2 signal transduction at a site distal to the cell surface binding site) and illustrate the importance of studies of receptor signaling pathways to complement radioligand binding.