Alternative Splicing and Endoproteolytic Processing Generate Tissue-specific Forms of Pituitary Peptidylglycine a-Amidating Monooxygenase (PAM)*

The pituitary is a rich source of peptidylglycine a- amidating monooxygenase (PAM). This bifunctional protein contains peptidylglycine a-hydroxylating monooxygenase (PHM) and peptidyl-a-hydroxyglycine a-amidating lyase (PAL) catalytic domains necessary for the two-step formation of a-amidated peptides from their peptidylglycine precursors. In addition to the four forms of PAM mRNA identified previously, three novel forms of PAM mRNA were identified by exam- ining anterior and neurointermediate pituitary cDNA libraries. None of the PAM cDNAs found in pituitary cDNA libraries contained exon A, the 315-nucleotide (nt) segment situated between the PHM and PAL domains and present in rPAM-1 but absent from rPAM- 2. Although mRNAs of the rPAM-3a and -3b type encode bifunctional PAM precursors, the proteins differ significantly. rPAM-3b lacks a 54-nt segment en- coding an 18-amino acid peptide predicted to occur in the cytoplasmic domain of this integral membrane pro- tein; rPAM-3a lacks a 204-nt segment including the transmembrane domain and encodes a soluble protein. rPAM-5 is identical to rPAM-1 through nt 1217 in the PHM domain; alternative

The pituitary is one of the richest sources of peptidylglycine a-amidating monooxygenase (PAM; EC 1.14.17.3)' in the adult rat (1,2). Immunocytochemical studies indicate that the highest levels of PAM protein are found in gonadotropes, but detectable levels of PAM protein are found in each of the major pituitary cell types (3). Although none of the major anterior pituitary hormones is a-amidated, several amidated peptides are synthesized in the anterior pituitary (4, 5). Following thyroidectomy, levels of PAM mRNA in the anterior pituitary rise severalfold, along with levels of the mRNAs encoding several a-amidated peptides (5,6). Intermediate pituitary melanotropes produce large amounts of two a-amidated products from proopiomelanocortin (a-melanocyte stimulating hormone and joining peptide) (4,7), and the major peptide products stored in the neural lobe (oxytocin and vasopressin) are a-amidated.
Peptide a-amidation involves a two-step reaction with a peptidyl-a-hydroxyglycine intermediate (8)(9)(10)(11)(12). The PAM precursor protein encodes both of the enzymatic activities involved in peptide a-amidation (13-15). The first enzyme, peptidylglycine a-hydroxylating monooxygenase (PHM), is contained within the NH2-terminal third of the rat PAM-1 precursor ( Fig. 1) and catalyzes the copper, molecular oxygen, and ascorbate-dependent formation of peptidyl-a-hydroxyglycine. The second enzyme, peptidyl-a-hydroxyglycine a-amidating lyase (PAL), follows the PHM domain and precedes the putative transmembrane domain; although spontaneous conversion of the a-hydroxyglycine intermediate into a-amidated product occurs at high pH, conversion at physiological pH values requires the action of PAL.
By screening an adult rat atrium cDNA library, we previously identified four forms of PAM mRNA that arise from the single copy PAM gene by alternative splicing (Fig. 1) (16 -18). PAM mRNAs of the rPAM-1 type are the longest; removal of exon A gives rise to rPAM-2, and removal of exons A and B gives rise to rPAM-3. In rPAM-4, a unique 3"region replaces the sequence of rPAM-1 following exon A; as a result, rPAM-4 encodes only the PHM domain. Based on Northern blot analysis, the anterior and neurointermediate lobes of the rat pituitary lack large amounts of rPAM-1 type mRNA and The abbreviations used are: PAM, peptidylglycine a-amidating monooxygenase; PHM, peptidylglycine a-hydroxylating monooxygenase; PAL, peptidyl-a-hydroxyglycine a-amidating lyase; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; nt, nucleotide(s); bp, base pair(s); kb, kilobase pair(s). I , e L y r C " " " " " . " " " " . " " " " " " " . . . " " " . " . . . . , YLyrClySer 2 7 9 1 2 9 9 6 rPAI1-la Iieiypl\---~pHIr*rp~~Ly.LeuCL~lerSerSerGlyArqV~iieuGiy~rqPhelrgClyLyrGlySer I ) T C I U L \ C M C C C C~~C C C~~~~~~~~~~~~~~~~~~~*~~~~~~~~~~~~~~~~~~~~~~~~C A~ rPAn-3Q i l e L y . C l u i i l a C ; u l l l . Y a l Y a l C l u P r o L y r Y I l C l h~~~~~~~G l " L~" G l " L y~~~~G l " C I I C L . 9~C I \ G M I I C T G I~~~~*~~~~~~~~~~~~~~~~~~~~~~~~G~T C T C~~~~~~~~C~~C T G G T r~r T C~~ C l " L y l C l n L y r L e " S e r I h r C l u P r o G l y S e r C l y V . r l I l P P r o 219s instead contain PAM mRNAs smaller than those found in the atrium (2). Reverse transcription coupled to the polymerase chain reaction using oligonucleotide primers spanning most of the protein coding region of rPAM-1 demonstrated that the forms of PAM mRNA in the anterior and neurointermediate lobes of the pituitary were similar to each other and included forms in addition to those characterized in adult atrium (18). By constructing and screening anterior and neurointermediate pituitary cDNA libraries, we were able to identify three additional novel forms of PAM mRNA.

C T G C T C C I C C r G C T C C C C
While rPAM-1 and -2 mRNA encode PAM proteins with a signal sequence and a putative transmembrane domain near their COOH terminus, the proteins encoded by rPAM-3 and -4 mRNA lack a transmembrane domain (17, 18). Despite a similar distribution of forms of PAM mRNA in the anterior and neurointermediate lobes of the pituitary, the majority of the PAM activity in the neurointermediate lobe is soluble, whereas equivalent amounts of soluble and membrane-associated PAM activity are found in the anterior pituitary (2,19). In this study, the major forms of PAM protein in the anterior and neurointermediate lobes of the pituitary were identified using antipeptide antibodies. In addition to tissuespecific alternative mRNA splicing, PAM proteins are subject to tissue-specific endoproteolytic processing.

MATERIALS AND METHODS AND RESULTS*
Identification of Novel rPAM cDNAs in Pituitary cDNA Libraries-cDNA libraries prepared from rat neurointermediate and anterior pituitary poly(A)+ RNA were screened with cDNA probes spanning the sequence of rPAM-1 (Fig. 1). Two novel types of PAM cDNA, rPAM-3a and rPAM-3b, were identified by characterizing PAM cDNA inserts from the neurointermediate pituitary library (Fig. 2). Inserts of each type were sequenced and found to represent additional splicing variants. cDNAs of the rPAM-3b type (Intl2) differed from rPAM-2 only by the deletion of a 54-nt segment corresponding to the final 54 nt of the 258-nt region referred to as exon B. cDNAs of the rPAM-3a type (Int22) differed from rPAM-2 only by the deletion of the first 204 nt of exon B.
Analysis of the gene encoding rat PAM indicates that the region previously referred to as exon B is composed of two exons: a 204-nt exon (exon B.) is situated approximately 3000 nt upstream of a 54-nt exon (exon Bb) (25). Exon Bh corre-* Portions of this paper (including "Materials and Methods," part of "Results," and Figs. 1, 4,5, 7, and 8) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. sponds exactly to the 54 nt deleted from bPAM-1 (originally called APAM-1) to form bPAM-2 (XPAM-5) (24).
The 853-amino acid rPAM-3b preproprotein has a predicted molecular weight of 94,721 (PI 5.76) and lacks an 18-amino acid peptide predicted to reside in the cytoplasmic domain of rPAM-2 (Fig. 2). The deleted 2115-dalton peptide (PI 10.83) is extremely hydrophilic and includes a pair of basic amino acids (Arg903-LysSo4; amino acid positions are all numbered as for the protein encoded by rPAM-1). The 803-amino acid rPAM-3a preproprotein has a predicted molecular weight of 89,344 (PI 5.78) and lacks a 68-amino acid peptide present in rPAM-1 and -2; the deleted peptide consists of a hydrophilic domain followed by the hydrophobic 24-amino acid putative transmembrane domain and the subsequent Arg-Trp-Lys-Lys894 putative stop transfer signal. This 7492-dalton peptide has a PI of 9.06 and lacks Cys residues or potential Nglycosylation sites. Thus mRNAs of the rPAM-3b type would be expected to encode an integral membrane protein, whereas mRNAs of the rPAM-3a type would be expected to encode a soluble protein.
When the PAM containing cDNA inserts in the anterior pituitary library were subsequently examined, a novel type of PAM cDNA (Ant67, rPAM-5 type) was identified by virtue of its failure to hybridize with the cDNA probe from the 3'third of rPAM-1 (Fig. 1). Upon sequence analysis, Ant67 was found to be identical to rPAM-1 from bp 247 through bp 1217 (bp 971 of Ant67) (Fig. 3). A novel 1.1-kb sequence followed; this relatively AT-rich region (61% AT) did not terminate with a poly(A) tail but contained four consensus poly(A) addition signals (26, 27). The novel 3'-domain of Ant67 bore no resemblance to the novel 3"domain of rPAM-4 (18). The point of divergence of Ant67 follows amino acid Gly307 of rPAM-1; Ant67 encodes an additional pentapeptide before an in-frame stop codon is reached.
The 312-amino acid preproprotein encoded by Ant67 has a predicted molecular weight of 34,674 and a PI of 8.93. Following removal of the signal peptide, the protein encoded by Ant67 would have a molecular weight of 32,151; the Lys-Arg35 sequence following the putative propeptide is the only paired basic amino acid sequence in the protein encoded by rPAM-5. Two cysteine residues and the -His-Gl~-His-His~~~ sequence postulated to play a role in the interaction of the PHM domain with copper are absent from the rPAM-5 protein. Transient expression of cDNA encoding rPAM-5 in human fibroblasts (hEK293) yielded expression of foreign protein but failed to yield PHM activity under the same conditions yielding PHM activity upon expression of cDNA encoding rPAM-4.
The presence of mRNAs of the rPAM-5 type in several tissues was demonstrated using reverse transcription and the polymerase chain reaction (Fig. 4). Levels of the amplified rPAM-5 specific product generated with cDNA from the various tissues mirrored total levels of PAM activity (2). Based on its prevalence in atrial and pituitary cDNA libraries, rPAM-5 is not a major transcript and attempts to visualize rPAM-5 mRNA on Northern blots were unsuccessful. To eliminate the possibility that Ant67 was the result of a cloning artifact, the structure of this region of the PAM gene was investigated using the polymerase chain reaction (Fig. 5). The PHM exon terminating with nt 1217 is separated from the exon containing the unique 3'-end of rPAM-5 by an approximately 550-nt i n t r~n .~ Thus mRNAs of the rPAM-5 type are the product of alternative splicing and are not generated by failure to remove the intron contiguous with nt 1217.
Comparison of PAM Proteins Found in Anterior and Neu-    Comparison of PAM proteins in atrial, anterior, and neurointermediate pituitary secretory granules by Western blot analysis. A, a crude secretory granule fraction was prepared from adult rat anterior and neurointermediate pituitary and from adult rat atrium by differential centrifugation. The atrial and anterior pituitary samples each contained 20 pmol/h PAM activity (8.4 and 19.6 pg of protein, respectively); the neurointermediate pituitary sample contained 4 pmol/h PAM activity (15 pg of protein). The samples were visualized with PAL antibody (Ab69). B and C, a more concentrated preparation of crude pituitary granules was analyzed. Each sample of anterior pituitary granules contained 100 pg of protein (1150 pmol/h PHM activity; 3100 pmol/h PAL activity). Each sample of neurointermediate pituitary granules contained 97 pg of protein (420 pmol/h PHM activity; 2900 pmol/h PAL activity). Following transfer to Immobilon-P, blots were exposed to the PAM antibodies indicated; exposure times were adjusted to give bands of comparable intensity. The exon B. antibody (Ab26) was applied to blots that had been stripped; this antibody cross-reacts with several lower molecular weight proteins not thought to be related to PAM. The location of molecular weight markers analyzed in a separate lane are indicated on the right; apparent molecular weights of the various PAM proteins are indicated on the left. rointermediate Pituitary-Despite the presence of similar forms of PAM mRNA in the anterior and neurointermediate lobes of the rat pituitary, previous studies indicated that membrane associated PAM was more prevalent in anterior pituitary than in neurointermediate pituitary extracts (2). The PAM proteins found in any tissue reflect both the forms of PAM mRNA present and the co-or post-translational modifications that occur. The higher molecular weight PAM proteins in secretory granule-enriched fractions prepared from rat anterior pituitary, neurointermediate pituitary, and atrium were compared by Western blot analysis (Fig. 6A). PAM proteins were visualized with affinity-purified rabbit polyclonal antibody to a peptide within the PAL catalytic domain. Granules from each tissue contained a distinctive collection of proteins recognized by the PAL antibody. As expected, atrial granules contained primarily intact rPAM-1 (120 kDa) and rPAM-2 (105 kDa) (28). Almost no protein the size of rPAM-1 was detected in anterior or neurointermediate pituitary granules. Both anterior and neurointermediate pituitary granules contained a protein the size of rPAM-2. A characteristic set of smaller PAL proteins (95, 84, and 75 kDa) were present in both anterior and neurointermediate pituitary granules. The 75-kDa PAL protein was a major component of neurointermediate pituitary granules, whereas the 105-kDa PAL protein was more prevalent in anterior pituitary granules.

G T A T N \ A A C G A C ? A T h G ? T T T T T T C C A G A A G M T~C T A A T A A G G C A T G T T T G A T G~A T T T T T A T T A T G A T G C
In order to investigate further the PAM proteins in pituitary granules, much more concentrated aliquots of a different preparation of anterior and neurointermediate pituitary granules were fractionated and visualized with antisera to synthetic peptides from within the PHM, PAL, and COOHterminal domains of rPAM-1 (Fig. 6, R and C). A complex pattern of PAM proteins was identified in secretory granules from both tissues. Although PAM proteins of similar apparent molecular weight were detected in both anterior and neurointermediate pituitary granules, different forms of PAM protein predominated in anterior and neurointermediate pituitary granules.
Anterior pituitary granules contain a set of PAM proteins cross-reactive with antisera to peptides within both the PHM and PAL catalytic domains (Fig. 6B). The most prominent bands had molecular masses of 105 k 3 kDa, 95 f 3 kDa, and 75 f 3 kDa; minor and somewhat variable amounts of an 84 f 3 kDa protein detected by PHM and PAL antisera were also present. In contrast, a 75 kDa PAM protein was the major PAM protein found in neurointermediate pituitary granules; only minor amounts of a 105-kDa PAM protein were present. The same set of higher molecular weight proteins were visualized by antiserum to a peptide closer to the NH, terminus of PHM (rPAM(116-131)) ( Fig. 6B). Although a number of smaller PHM and PAL proteins were visualized in secretory granules from both regions of the pituitary, no proteins smaller than 75 kDa were visualized by antisera to both PHM and PAL (Fig. 6B). PHM proteins of 44-45 kDa were visualized with varying intensity by both PHM antisera. The 42-kDa protein visualized by antibody to rPAM(116-131) was not visualized as well by antibody to rPAM(293-315); the 59-kDa protein visualized by antibody to rPAM(293-315) was not visualized by the other PHM antibody, and its relationship to PAM is unclear. Small amounts of PAL proteins of approximately 50 kDa were visualized in both anterior and neurointermediate pituitary granules.
In order to aid in identification of the various PAM proteins, the same samples were visualized with antisera to peptides contained within exon B, and the COOH-terminal cytoplasmic domain of rPAM-1 (Fig. 6C). Antibody to exon B, visualized the 105-kDa PAM protein, but not the 75-kDa PAM protein in the anterior pituitary granule preparation; adequate evaluation of the cross-reactivity of the 95-kDa PAM protein(s) requires separation of soluble and membrane proteins. The COOH-terminal domain antibody detected 105and 95-kDa PAM proteins, but not the 75-kDa PAM protein.
Since all of the PAM mRNAs encoding both PHM and PAL also encode this COOH-terminal determinant, the 75-kDa PAM protein must arise from precursor forms of PAM (rPAM-1, -2, -3a, -3b, or -3) by endoproteolytic cleavage. In the neurointermediate pituitary granules, an approximately 28-kDa protein was visualized by the COOH-terminal domain antiserum; it is not yet clear whether this protein is derived from PAM.
The 105-kDa PAM protein is found in anterior pituitary membranes washed with 0.1 M Na2C03 to remove peripheral proteins and is thought to represent rPAM-2 and -3b (Fig. 7). Small amounts of a 95-kDa PAM protein lacking COOHterminal antigenic determinants are also found associated with the membranes and may represent a processed form of these proteins. Neurointermediate pituitary membranes also contain small amounts of a 105-kDa PAM protein (Fig. 8B). The soluble fraction of anterior pituitary secretory granules contains large amounts of both the 75-kDa PAM protein recognized by antisera to PHM and PAL and the monofunctional 44-45 kDa PHM protein (Fig. 8A). The 95-kDa PAM protein found in the soluble fraction is recognized by antisera to the COOH-terminal domain and is thought to represent intact rPAM-3/3a. Very little monofunctional PAL protein is found in the soluble fraction of anterior pituitary granules.

DISCUSSION
A single complex gene encodes PAM in the rat (25, 29). The functional consequences of expressing the seven different forms of PAM mRNA identified in the Sprague-Dawley rat (16-18) are significant. Five of the mRNAs encode bifunctional PAM proteins, with three of the mRNAs encoding PAM proteins with a transmembrane domain and two encoding soluble bifunctional proteins (Fig. 9). Rat PAM-4 mRNA encodes a soluble form of PHM (18). Rat PAM-5 mRNA encodes only part of the PHM domain and current studies indicate that rPAM-5 is inactive; this observation is consistent with the fact that the protein encoded by rPAM-5 does not include the entire region of homology to dopamine pmonooxygenase (30). The importance of synthesizing an inactive truncated PHM protein is unclear; alternative splicing generates an inactive form of glutamic acid decarboxylase that is expressed at high levels early in embryonic brain development (31).
We previously demonstrated the tissue-specific expression of different forms of PAM mRNA, thus atrium contains primarily rPAM-1 and -2 mRNA, whereas little rPAM-1 mRNA is found in the pituitary (2,18). In this study, the anterior and neurointermediate lobes of the Sprague-Dawley rat pituitary were found to contain a very similar collection of PAM mRNAs; mRNAs of the rPAM-2 and -3b type were the most prevalent, with less rPAM-3, -3a, and -1 and very small amounts of rPAM-4 and -5. Despite the presence of similar forms of PAM mRNA, the PAM proteins found in the anterior and neurointermediate lobes of the pituitary differ. Thus both alternative splicing and post-translational processing contribute to the tissue specific production of proteins derived from PAM.
When exon A is absent, no paired basic potential endoproteolytic cleavage site separates the PHM and PAL domains. Exons B. and Bb separate the PAL catalytic domain from the COOH-terminal region; this COOH-terminal region forms the cytoplasmic domain of rPAM-1 and -2. Exon B, contains the transmembrane domain and its stop transfer signal, whereas exon Bb contains a pair of basic amino acids (Argso3-Lysgo4). The fact that exon Bb corresponds exactly to the 54-nt region distinguishing two forms of bovine PAM mRNA (24)  , and potential paired basic amino acid endoproteolytic cleavage sites (K = Lys, R = Arg) and N-glycosylation sites (irregular enclosed shape) indicated. Predicted molecular weights and isoelectric points (PI) of the nonglycosylated proproteins are shown. Arrows, endoproteolytic cleavage site thought to be used in the production of the 75-kDa PAM protein from rPAM-2, -3b, -3a, and -3. by exon BI, could perform different functions when expressed as part of rPAM-2 and rPAM-3a. In rPAM-2 this peptide forms part of the cytoplasmic domain and might affect intracellular routing of PAM. For example, the ligand-mediated internalization of the FcR,, receptor is governed by the presence or absence of a 47-amino segment in its COOH-terminal domain (32). In rPAM-3a the exon Bt, peptide should be situated within the secretory granule; in this location it could serve as a paired basic endoproteolytic processing site.
Several other laboratories have characterized PAM mRNAs from other tissues or other species. Type A and Type B PAM cDNAs were isolated from a rat medullary thyroid carcinoma cDNA library (33); except for minor differences, the type A cDNA is identical to rPAM-3b (it lacks the 315 bp of exon A and the 54 bp of exon Bb). The Type B cDNA is essentially identical to rPAM-1 until a point close to the 3'-end of exon B,; a 3-bp insertion is followed by 47 bp of exon Bb. The sequence of the Type B cDNA then diverges completely from rPAM-1; a stop codon is reached 55 amino acids after the transmembrane domain and the 3"untranslated region is extremely purine-rich (33). No PAM cDNAs with a 3'-end resembling that of Type B PAM cDNA were identified in the PAM cDNAs examined from our atrial and pituitary libraries.
Five types of PAM cDNA were identified in libraries prepared from the pituitaries of adult Wistar rats (29); the five forms arise via alternative splicing at the regions referred to in this study as exons A, B,, and Bb. Unexpectedly, different forms of PAM mRNA were found to be prevalent in Sprague-Dawley and Wistar rat pituitaries. As found previously in bovine pituitary (24), PAM cDNAs retaining exon A were prevalent in libraries prepared from Wistar rat pituitary (29); in contrast, PAM cDNAs retaining exon A were rare in libraries prepared from Sprague-Dawley pituitary. The scarcity of PAM mRNAs of the rPAM-1 type in Sprague-Dawley rat pituitary was confirmed by Northern blot analysis (2), reverse transcription coupled with polymerase chain reaction (18), and Western blot analysis of the PAM proteins present (Figs. 6 and 7). Differences between Sprague-Dawley and Wistar rats were not confined to the exon A region. Using an RNase protection assay the most prevalent form of PAM mRNA in the Wistar rat pituitary was found to lack exon B, and retain exon Bh (29); in the Sprague-Dawley rat this was the least prevalent splicing pattern. Forms of PAM mRNA retaining exon B, and lacking exon Bb were prevalent in the Sprague-Dawley rat and rare in the Wistar rat. Although PAM mRNAs encoding the transmembrane domain predominate in Sprague-Dawley rat pituitary (rPAM-2 and -3b), the major form of PAM mRNA in the Wistar rat pituitary lacks a transmembrane domain (29). Although we searched for forms of PAM mRNA retaining exon A and lacking exon B, and/or exon Bb, none were found. Comparison of the genes encoding PAM in Sprague-Dawley and Wistar rats may clarify the reasons for these differences.
Although PHM and PAL are active as independent soluble enzymes, and also active when part of a bifunctional membrane-associated protein (28), secretion of PHM and PAL along with the secretory granule content requires an endoproteolytic cleavage to separate the PAL domain from the transmembrane domain in forms retaining exon B,. Secretion of the proteins encoded by rPAM-3, -3a, -4, and -5 requires no endoproteolytic cleavages subsequent to signal peptide removal.
PAM undergoes tissue-specific endoproteolytic processing, with more extensive endoproteolytic processing of PAM occurring in the neurointermediate lobe. The only major integral membrane protein form of PAM seen in the anterior pituitary is a 105-kDa doublet likely to represent rPAM-2 and -3b; very little of this 105-kDa PAM protein is found in the neurointermediate lobe. Small amounts of a 95-kDa PAM protein detected in anterior pituitary membranes appear to lack at least part of the COOH-terminal cytoplasmic domain. The 95-kDa PAM protein found in the soluble fraction is visualized by antisera to the COOH-terminal domain of rPAM-1 and is likely a combination of rPAM-3/3a. The major soluble 75-kDa PAM protein observed in both anterior and neurointermediate pituitary granules lacks antigenic determinants for exon B, and the COOH terminus of rPAM-1 and could be derived from rPAM-2, -3, -3a, or -3b by endoproteolytic cleavage at one of the two paired basic amino acid sequences following the PAL domain. In CA-77 cells, processing at Lys-Lyse2* is thought to generate a soluble 75-kDa PAM protein from rPAM-3b (Type A); the PAM protein containing the peptide encoded by exon A (Type B mRNA) is cleaved to form 41-and 43-kDa proteins (33, 34). If endoproteolytic processing events generate the 58-and 44-45-kDa PHM products from rPAM-2, -3, -3a, or -3b, the cleavages must occur at nonpaired basic sites. Alternatively, the smaller PHM proteins could represent products of rPAM-4 and rPAM-5 mRNAs. No major membrane-associated processing products were detected with antisera to exon B, or the COOH-terminal domain of rPAM-1. Biosynthetic labeling of cells expressing individual forms of PAM mRNA will be required to delineate the steps involved in processing. We now have information on the endoproteolytic processing of PAM in several tissues. In the atrium, both PAM and pro-atrial natriuretic factor are stored in a largely unprocessed form; primary cultures of neonatal atrial myocytes cleave proANF at the time of secretion and also secrete large amounts of PHM and PAL activity, indicating that endoproteolytic cleavage of the PAM precursor must occur (35, 36). The endoproteolytic processing of PAM in the neurointermediate lobe of the pituitary is more extensive than that observed in the anterior lobe. The endoproteolytic processing of proopiomelanocortin is also much more extensive in the intermediate than in the anterior lobe of the pituitary. Given that the extent of PAM processing correlates with the extent of endogenous preprohormone processing, one wonders whether similar endoproteases are involved in processing PAM and its prohormone substrates. The tissue-specific endoproteolytic processing of PAM resembles the tissue-specific processing of prohormones; instead of generating sets of bioactive peptides, various forms of active enzyme are created. """_. eolvmeram chalu%?d!Qn. PCR was utllked to characterin the cDNA Inseris In phage or plasmlds and to evaluate the represenMIon 01 lorms 01 PAM cDNA In the llbrarlea Allquota 01 phage were heat denatured (5 mln, %? and wbjected to x ) ~ 25 wlw 01 PCR uslng 1 pH primer llmes r a n m from 2 mln lor plecsa smaller thsn 1 kb to 4 mln lor Me HItlre In& Products were (all were 17-mem) a0 described (is). Annealing temperalures were generally 52" and extension analyzed aner YIwalMIon with athldlum bromlde or after hanster to Nyiran and hybrldl2atlon wkh appropriate random primed cDNA pmbea In& Ienglh was datermlmd using T, and T, prlmsn and the torms of rPAM cDNA were determlned with prlmem spannlng exons A end B (18). cDNA was synthsslled Imm 2 . 5 pg total RNA uslng 1 pg ollgo(dT),,., (Phermacla) es primer and AMV Revem Transcriptase (Ute Sclenm) a0 descrlbed prevlously (IS). The amount. of cDNA from dlerent t l m e s utlllled for PCR were adjusted lo yield slgnals 01 comparable lntensitv (2).

A
Anterior pltultary granules Delinltlve idedlllcatlon 01 these proteins will require purillcation and sequence pituitary extracts were compared (Fig. 88). Like anterior pltultary membranes, neurointermediate pituitary membranes conlainad a 105 kDa protein detected by antisera to PHM and PAL: some 75 kDa PAM proteln along with a 53 kDa protein detected by this PHM antiserum remained membrane associated. The major bitunctlonal PAM protein In the soluble lraction was 75 kDa: lesser