Novel Alternatively Spliced Exon in the Extracellular Ligand-binding Domain of the Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) Type 1 Receptor (PAC1R) Selectively Increases Ligand Affinity and Alters Signal Transduction Coupling during Spermatogenesis

Abstract

The expression of the paracrine signaling hormone pituitary adenylate cyclase-activating polypeptide (PACAP) is regulated in a cyclical fashion during the 12-day spermatogenic cycle of the adult rat testis. The precise functions of PACAP in the development of germ cells are uncertain, but cycle-and stage-specific expression may augment cAMP-regulated gene expression in germ cells and associated Sertoli cells. Here we report the existence of a heretofore unrecognized exon in the extracellular domain of the PACAP type 1 receptor (PAC1R) that is alternatively spliced during the spermatogenic cycle in the rat testis. This splice variant encodes a full-length receptor with the insertion of an additional 72 base pairs encoding 24 amino acids (exon 3a) between coding exons 3 and 4. The PAC1R(3a) mRNA is preferentially detected in seminiferous tubules and is expressed at the highest levels in round spermatids and Sertoli cells. Analyses of ligand binding and signaling functions in stably transfected HEK293 cells expressing the two receptor isoforms reveals a 6-fold increase in the affinity of the PAC1R(3a) to bind PACAP-38, and alterations in its coupling to both cAMP and inositol phosphate signaling pathways relative to the wild type PAC1R. These findings suggest that the extracellular region between coding exons 3 and 6 of PAC1R may play an important role in the regulation of the relative ligand affinities and the relative coupling to G s (cAMP) and G q (inositol phosphates) signal transduction pathways during spermatogenesis.

Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: 20 Years after the Discovery

“…In the human and rat placenta, PAC1-R mRNA colocalizes with PACAP mRNA, and the two transcripts exhibit the same kinetics of expression throughout pregnancy (Koh et al, 2003(Koh et al, , 2005. In the testis, type I PACAP binding sites are found in germ cells (Shivers et al, 1991), Leydig cells (Romanelli et al, 1997), and Sertoli cells (Heindel et al, 1992;Daniel et al, 2001). In situ hybridization and Northern blot analyses indicate that PAC1-R and VPAC2-R mRNAs, but not VPAC1-R mRNA, are expressed in germ cells (Usdin et al, 1994;Krempels et al, 1995;El-Gehani et al, 1998a,b;Koh and Won, 2006).…”

Duplicated receptors for VIP and PACAP (VPAC1R and PAC1R) in a teleost fish, Fugu rubripes

“…VPAC 1 R is found mainly in the cerebral cortex, hippocampus, pineal gland, lungs and intestines and VPAC 2 R, which is proposed to be the neuro-endocrine receptor for VIP, is found in the hypothalamus, pituitary, adrenal glands, pancreatic islets, testes and ovaries (Usdin et al 1994, Vaudry et al 2000a. In mammals, PAC 1 R is expressed predominantly in the CNS, however the situation is more complex, with the production of alternative splice variants (hip-hop cassettes) which influence receptor selectivity for the PACAP38 and PACAP27 ligand isoforms (Spengler et al 1993, Journot et al 1994, Pantaloni et al 1996, Chatterjee et al 1997, Daniel et al 1998, Dautzenberg et al 1999. Isolation and functional characterisation of the PAC 1 R in goldfish indicates that the PACAP ligand acts as a novel GH-releasing factor in this organism (Wong et al 2000).…”

Stimulation of the ERK Pathway by GTP-loaded Rap1 Requires the Concomitant Activation of Ras, Protein Kinase C, and Protein Kinase A in Neuronal Cells

“…PACAP neurotrophic activity is mediated by the activation of Pac1, the PACAP-specific GPCR. Pac1 displays a complex pattern of alternative splicing that modulates its ligand binding and signaling properties (31)(32)(33)(34) resulting in the modulation of PACAP physiological effects (28,35). We and others (21,31) showed that Pac1 potently stimulated the AC and PLC activities as well as the ERK pathway that was implicated in its neurotrophic activity (23,36).…”