Pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, have been shown to be linked to adenylate cyclase in several cell types and cell lines. PACAP specific binding sites were designated type 1 PACAP receptors; receptors shared with VIP were named type II PACAP receptors.
Classification of PACAP receptors
PACAP receptors are classified according to their relative affinities to PACAP and VIP. Two types of PACAP binding sites and three receptor subtypes have been identified. PACAP type I receptors (PACI-R) show higher affinity (100 to 1000-fold) to PACAP than VIP; however, PACAP type II receptors exhibit equal affinity to PACAP and VIP. Type II receptors can further be divided into 2 subtypes, VPACI-R and VPAC2-R, based on their differential binding affinities to secretin and helodermin, which is a 35-amino acid peptide isolated from lizard venom. VPACI-R, traditionally regarded as the classical VIP receptor, has low affinity binding for secretin. On the other hand, VPAC2-R, has no affinity for secretin but has higher affinity for helodermin than for PACAP and VIP. Analysis of [125I]PACAP27 binding on membrane preparations from various tissues indicated that all PACAPNIP receptors are widely distributed in the CNS as well as the peripheral tissues such as the endocrine organs, pituitary, adrenal gland, and ovary, again suggesting that PACAP is involved in diverse physiological functions.
Expression of PACAP receptors
Between July and September 1993, six laboratories independently reported the cloning of cDNAs encoding for the rat PAC1 receptor and the deduced amino acid sequences of the PAC1 receptor and its subtypes. Subsequently, Ogi et al cloned the cDNA for the human PAC1 receptor, and Miyamoto et al described the cDNA for the bovine PAC1 receptor. These studies showed that the PACAP receptor belongs to the secretin/VIP receptor family with conserved seven transmembrane domains and contains many of the motifs expected for a G protein-coupled receptor. The cDNAs were expressed in mammalian cells, and these cells were found to bind PACAP specifically and to increase intracellular cAMP in response to PACAP. While the N-terminal domains of the secretin/VIP receptor family are highly divergent, they have substantial similarity at specific amino acid positions, including the six cysteine residues postulated to confer a general conformation to the extracellular ligand-binding domains of these receptors. The amino acid sequence of PACAP is most similar to VIP and the PAC1 receptor has the highest degree of similarity to the VIP receptor (51%). The PACAP receptor exhibits also similarity to the receptors for calcitonin (31%), parathyroid hormone (37%), glucagon (38%), glucagon-like peptide (37%), GHRH (41%), and secretin (47%). Inagaki et al cloned a cDNA encoding a third PACAP receptor (VPAC2-R) from the mouse insulin-secreting beta-cell line MIN6. The receptor expressed in mammalian cells with the cDNA showed a similar binding affinity for VIP and PACAP, and a similar increase in intracellular cAMP after stimulation with VIP or PACAP.
Eight subtypes of the PAC1-R that result from alternative splicing have been described. Among the PAC1-R subtypes, six of the variants differ from one another by the absence or presence oftwo cassettes named "hip" and "hop", possibly inserted at the end of the third intracellular loop of the receptor. The resulting variants have been named PAC1-Rs (short receptor without either cassette), PAC1-R-hip, PAC1-R-hopl, PAC1-R-hop2, PAC1-R-hiphopl, and PAC1-R-hiphop2. Both the short form and the hop variants potently activate AC and PLC, whereas the hip variant does not stimulate PLC. The hip-hop variants display an intermediate signal transduction pattern with an altered ability to activate PLC. More recently, another splice variant of the PAC1-R, characterized by a 21 amino acid deletion in the N-terminal extracellular domain, was cloned. Potencies for the stimulation of PLC by PACAP38 were approximately 10 times greater than by PACAP27 for PACI-R-s, whereas the PLC stimulating potencies of PACAP38 and PACAP27 were similar for PAC1- R-vs. The extracellular 21-amino acid domain may modulate receptor selectivity with respect to PACAP38 and PACAP27 and control the relative potencies of the two PACAPs for stimulation of PLC. Another distinct variant of PAC1-R-s differs primarily by discrete sequences in transmembrane domains II and IV. This variant was designated PAC1-R-TM4. Unlike the other PAC1-R variants, PAC1- R-TM4 activates neither AC nor PLC in response to PACAP in transient or stable expression systems. PACAP stimulates increases in [Ca2+]i in cells expressing PACI-R-TM4 by activating L-type Ca2+ channels, a response not elicited by stimulation with VIP. The signal phenotype of PAC1-R-TM4 is involved in the regulation of insulin secretion from pancreatic beta-cells. Coexpression of the mRNA for PAC1-R-TM4 and other PAC1-R splice variants was observed in the cerebellum, cerebral cortex and brainstem. In contrast, the mRNA for PAC1-R-TM4 was not detected in the spinal cord where transcripts encoding other splice variants of PAC1-R were clearly expressed.
Kostrzewa, R. M., Archer, T., & Sweden, G. Current Topics in Neurotoxicity.