Testes Exhibit Elevated Expression of Calcitonin Gene-Related Peptide Receptor Component Protein

0013-7227/99/$03.00/0 Endocrinology Copyright © 1999 by The Endocrine Society Vol. 140, No. 3 Printed in U.S.A. Testes Exhibit Elevated Expression of Calcitonin GeneRelated Peptide Receptor Component Protein* WAYNE BALKAN, EDWARD L. OATES, GUY A. HOWARD, AND BERNARD A. ROOS Geriatric Research, Education, and Clinical Center, Veterans Affairs Medical Center, and the Departments of Medicine (W.B., E.L.O., G.A.H., B.A.R.), Biochemistry and Molecular Biology (G.A.H.), and Neurology (B.A.R.), University of Miami School of Medicine, Miami, Florida 33101 ABSTRACT Calcitonin gene-related peptide (CGRP) receptor component protein (RCP) is a novel protein that modulates CGRP responsiveness in a variety of cell types. Using probes based on the isolation of CGRPRCP complementary DNA (cDNA) from a guinea pig organ of Corti cDNA library, we cloned human (h) and mouse (m) CGRP-RCP cDNAs, both of which encode 148-residue proteins that at the amino acid levels are approximately 88% identical to each other and to the 146-residue guinea pig CGRP-RCP. Northern blot analysis confirmed the presence of CGRP-RCP messenger RNA in all of the human and mouse tissues tested. In these human tissues, hCGRP-RCP messenger RNA (major band at ;3.1 kb, minor band at ;7.5 kb) was most prevalent in the testis. In the mouse, the highest abundance of CGRPRCP RNA was clearly in the testis (major band at ;1.6 kb, minor band C ALCITONIN gene-related peptide (CGRP) is a 37amino acid neuropeptide with widespread expression and a wide array of biological effects (see Refs. 1–3 for reviews), including neuromodulation (3, 4), vasodilation (5), and bone anabolism (6 –9). In addition, a role for CGRP in reproduction is becoming increasingly evident. CGRP influences many aspects of mammalian development (10 –13), affects the function of male (14 –18) and female (19 –23) reproductive tissues, and plays a critical role during parturition (19 –21). The major cellular response to CGRP is an increase in the levels of intracellular cAMP (1–3). This response inspired an expression-cloning strategy based on the Xenopus laevis oocyte expression system, which led to the initial identification of the CGRP receptor component protein (CGRP-RCP) by its ability to confer CGRP responsiveness to these cells (24). A similar cloning strategy was recently used to identify the receptor activity-modifying proteins (RAMPs), a family of proteins that affect the membrane presentation, glycosylation, and ligand specificity of the calcitonin receptor-like receptor (a CGRP receptor) and the endogenous oocyte CGRP receptor (25). Although the exact relationship among CGRP-RCP, CGRP receptors, and RAMPs has not been eluReceived July 15, 1998. Address all correspondence and requests for reprints to: Wayne Balkan, Ph.D., Veterans Affairs Medical Center, 1201 NW 16th Street, Geriatric Research, Education, and Clinical Center (11GRC), Miami, Florida 33125. E-mail: . * This work was supported in part by the Department of Veterans Affairs. Part of this work was conducted during the tenure of an Initial Investigatorship Award (to W.B.) from the American Heart Association, Florida Affiliate, Inc. at ;1.1 kb). Based on this tissue distribution of RNA, we sought to identify the cells in the murine testis that contained CGRP-RCP protein. Numerous antisera generated against hCGRP-RCP, including one to recombinant hCGRP-RCP, exhibited strong immunoreactivity localized to the head region of spermatozoa. No CGRP-RCP immunoreactivity was observed in other cells at less mature stages of sperm maturation, in Sertoli or interstitial (Leydig) cells, or in human spermatozoa. Murine epididymal (mature) spermatozoa exhibited CGRP-RCP immunoreactivity identical to that of testicular spermatozoa. Spermatozoa that underwent an experimentally induced acrosome reaction (acrosomal discharge) lost their CGRP-RCP immunoreactivity. Therefore, it appears that CGRP-RCP is associated with the acrosome, suggesting that it may play an important role in reproduction. (Endocrinology 140: 1459 –1469, 1999) cidated, the discovery of these two accessory proteins illustrates the complexity associated with CGRP responsiveness. Our initial cloning of the complementary DNA (cDNA) for CGRP-RCP was from a guinea pig organ of Corti cDNA library (24). Unlike the reported CGRP receptors (26, 27) that belong to the family of seven-transmembrane-spanning, G protein-coupled receptors, or RAMPs (25), CGRP-RCP has no obvious membrane-spanning domain. Its structure suggests that rather than directly binding CGRP, this factor, in combination with the endogenous CGRP receptors present in the Xenopus oocyte (28), enables the oocytes to respond to CGRP via a stimulation of cAMP and/or an increase in protein kinase A activity (24). Additional evidence that CGRP-RCP functions in CGRP signaling derives from two observations. First, cells of the guinea pig cerebellum and cochlea that synapse with CGRP-containing neurons and presumably contain CGRP receptors also contain CGRP-RCP mRNA by in situ hybridization (24). Second, a functional assay in murine uterus established a correlation between the presence of CGRP-RCP and CGRP responsiveness (19). When we began this work, there was little information on the tissue distribution of CGRP-RCP despite virtually every tissue being a target for CGRP (1–3). Therefore, in seeking more relevant and convenient models for studying CGRPRCP, we cloned human and mouse versions of CGRP-RCP and ascertained their tissue distributions. We found that human (h) and mouse (m) CGRP-RCP are highly homologous to each other and to the guinea pig (gp) CGRP-RCP at the amino acid level and that they were expressed in all tissues examined. Particularly striking were the high levels of expression in murine testis, where our CGRP-RCP antisera reacted strongly with the head region of spermatozoa, spe- 1459 1460 CGRP-RCP IN MURINE ACROSOMES cifically in the acrosome. Although the roles of CGRP-RCP and CGRP in the functioning of spermatozoa are not understood, the high concentration of CGRP-RCP in acrosomes of murine spermatozoa suggests that this protein plays an important function in reproduction. Materials and Methods Screening of cDNA libraries Endo • 1999 Vol 140 • No 3 13.5- and 14.5-day postcoitum (dpc) total fetal cDNA library was highly homologous to the mouse and human clones we had isolated. Plasmid DNA from this clone (GenBank accession no. W99936; American Type Culture Collection, Manassas, VA) was isolated using the Qiagen plasmid isolation kit (Qiagen, Valencia, CA), sequenced (GenBank accession no. AF118271), and subcloned into the pcDNA3 vector (Invitrogen, San Diego, CA). Computer comparisons to genetic database sequences The Wisconsin GCG package of programs (Wisconsin Package Versions to 9.1, Genetics Computer Group, Madison, WI) was used for DNA sequencing (FAS) and DNA and amino acid database similarity searching and motif matching (fasta, blast, motifs, profile (...truncated)