Breeding Stock-Specific Variation in Peptidylglycineα-Amidating Monooxygenase Messenger Ribonucleic Acid Splicing in Rat Pituitary

0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society Vol. 141, No. 2 Printed in U.S.A. Breeding Stock-Specific Variation in Peptidylglycine ␣-Amidating Monooxygenase Messenger Ribonucleic Acid Splicing in Rat Pituitary* GIUSEPPE D. CICCOTOSTO, TRACEY A. HAND, RICHARD E. MAINS, AND BETTY A. EIPPER Departments of Neuroscience and Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 ABSTRACT Peptidylglycine ␣-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the carboxyl-terminal amidation of glycine-extended peptides in a two-step reaction involving a monooxygenase and a lyase. Several forms of PAM messenger RNA result from alternative splicing of the single copy PAM gene. The presence of alternately spliced exon A between the two enzymatic domains allows endoproteolytic cleavage to occur in selected tissues, generating soluble monooxygenase and membrane lyase from integral membrane PAM. While using an exon A antiserum, we made the unexpected observation that Charles River Sprague Dawley rats expressed forms of PAM containing exon A in their pituitaries, whereas Harlan Sprague Dawley rats did not. Forms of PAM containing exon A were expressed in the atrium and hypothalamus of both types of Sprague Dawley rat, although in different proportions. PAM transmembrane domain splicing also differed between rat breeders, and full-length PAM-1 was not prevalent in the anterior pituitary of either type of rat. Despite striking differences in PAM splicing, no differences in levels of monooxygenase or lyase activity were observed in tissue or serum samples. The splicing patterns of other alternatively spliced genes, pituitary adenylate cyclase-activating polypeptide receptor type 1 and cardiac troponin T, did not vary with rat breeder. Strain-specific variations in the splicing of transcripts such as PAM must be taken into account in analyzing the resultant proteins, and knowledge of these differences should identify variations with functional significance. (Endocrinology 141: 476 – 486, 2000) P EPTIDYLGLYCINE ␣-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the carboxyl-terminal amidation of glycine-extended peptides in a two-step process (1). Peptidylglycine ␣-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction, and peptidyl-␣-hydroxyglycine ␣-amidating lyase (PAL) catalyzes the second step. Several forms of PAM messenger RNA (mRNA) result from alternative splicing of the single copy rat PAM gene (2– 4) (Fig. 1). PAM-1, the longest form, is composed of a signal and propeptide sequence followed by the PHM catalytic domain, a noncatalytic domain referred to as exon A (exon 16), the PAL catalytic domain, a transmembrane domain, and a carboxyl-terminal domain (1). Removal of exon A gives rise to PAM-2 and removal of exons A, Ba, and Bb gives rise to PAM-3. Splicing of the rat PAM gene is tissue specific and developmentally regulated (5, 6). In addition to alternative splicing, tissue-specific endoproteolytic cleavage of the various PAM proteins generates a wide variety of protein products (3, 6). In the rat, the presence of exon A allows endoproteolytic cleavage to occur in selected tissues, generating soluble PHM and membrane PAL from PAM-1; secretion of soluble PHM occurs along with secretion of peptide products (7). Lacking exon A, the bifunctional PAM-2 protein remains intact and membrane associated unless endoproteolytic cleavage separates PAL from the transmembrane domain. The functional significance of exon A is supported by the fact that transcripts with and without exon A have also been identified in human and Lymnaea (8 –10). Although Lymnaea exon A shares only 8% sequence identity with rat exon A, it does have a Lys-Lys sequence that could constitute an endoproteolytic cleavage site (8). In Lymnaea, single cell PCR has been used to demonstrate that expression of PAM is limited to neurons producing amidated peptides, and that expression of PAM transcripts including exon A is further limited to a subset of these neurons. During rat development, the only PAM transcripts in the ependymal region of the spinal cord and the ventricular zone of the hippocampus lack exon A, again suggesting a functional role for this exon (11). We used recombinant rat exon A to develop a rabbit polyclonal antiserum specific for forms of PAM that include this region. The polyclonal antiserum recognizes sites in both the PHM and PAL products generated from PAM-1 (12). In the process of using this antiserum, we made the unexpected observation that Sprague Dawley rats purchased from one breeder expressed forms of PAM containing exon A in their anterior pituitary and neurointermediate lobe, while Sprague Dawley rats obtained from a different breeder did not. A number of reports describe substantial differences in experimental observations when using Sprague Dawley rats supplied by different breeders. Marked differences were observed in the projections from locus coeruleus neurons to the spinal cord when Sprague Dawley rats from different breed- Received August 4, 1999. Address all correspondence and requests for reprints to: Dr. Betty A. Eipper, Department of Neuroscience, WBSB 907, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205. E-mail: . * This work was supported by NIH Grant DK-32949 and Human Frontiers Science Program Organization Fellowship LT-647/97 (to G.D.C.). 476 PITUITARY PAM EXPRESSION DIFFERS BETWEEN RAT SUPPLIERS 477 (20 ␮g protein) were fractionated on either 10% or 12% polyacrylamide, 0.25% N,N⬘-methylene-bis-acrylamide/SDS gels, transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA), and visualized using the enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Arlington Heights, IL) (7, 22). Polyclonal rabbit antisera were raised against different regions of PAM: Ab JH1764 [rPAM-1-(37–382)] was used to detect PHM (7), Ab JH471 [rPAM-1-(464 – 864)] was used to detect PAL (7), Ab JH629 [rPAM-1-(409 – 497)]) was used to detect exon A (12), and Ab JH571 [rPAM-1-(898 –976)] was used to detect the carboxyl-terminal domain of PAM (22). PHM and PAL enzyme assays were performed in duplicate on serial dilutions of tissue extracts and serum as previously described, using ␣-N-acetyl-Tyr-Val-Gly and ␣-N-acetylTyr-Val-␣-hydroxyglycine as substrates, respectively (23). RNA extraction and PCR analysis FIG. 1. PAM proteins and their cleaved products. PAM-1 is the largest PAM protein, and all amino acids are numbered as in rat PAM-1: PHM domain (36 –392), PAL domain (498 – 831), and exon A (393– 497). Cleavage within exon A occurs at Lys-Lys437, resulting in soluble PHM and membrane-bound PAL-CD. Exon B (832–917) contains alternatively spliced exons Ba (832– 899) and Bb (900 –917). PAM-1s, PAM-2s, and PALs are the products formed from cleavage between PAL and the transmembrane domain (TMD). The COOH-termin (...truncated)