Breeding stock-specific variation in peptidylglycine alpha-amidating monooxygenase messenger ribonucleic acid splicing in rat pituitary.

Peptidylglycine alpha-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.

Kalirin inhibition of inducible nitric-oxide synthase.

Nitric oxide (NO) acts as a neurotransmitter. However, excess NO produced from neuronal NO synthase (nNOS) or inducible NOS (iNOS) during inflammation of the central nervous system can be neurotoxic, disrupting neurotransmitter and hormone production and killing neurons. A screen of a hippocampal cDNA library showed that a unique region of the iNOS protein interacts with Kalirin, previously identified as an interactor with a secretory granule peptide biosynthetic enzyme. Kalirin associates with iNOS in vitro and in vivo and inhibits iNOS activity by preventing the formation of iNOS homodimers. Expression of exogenous Kalirin in pituitary cells dramatically reduces iNOS inhibition of ACTH secretion. Thus Kalirin may play a neuroprotective role during inflammation of the central nervous system by inhibiting iNOS activity.

Kalirin, a multifunctional PAM COOH-terminal domain interactor protein, affects cytoskeletal organization and ACTH secretion from AtT-20 cells.

The production and regulated secretion of bioactive peptides require a series of lumenal enzymes to convert inactive precursors into bioactive peptides plus several cytosolic proteins to govern granule formation, maturation, translocation, and exocytosis. Peptidylglycine alpha-amidating monooxygenase (PAM), an enzyme essential for biosynthesis of many peptides, is an integral membrane protein with trafficking information in both its lumenal and cytosolic domains. Kalirin, a PAM cytosolic domain interactor protein with spectrin-like repeats and GDP/GTP exchange factor activity for Rac1, is expressed with PAM in neurons but is not expressed in the anterior pituitary or AtT-20 corticotrope cells. Expression of Kalirin alters the cytoskeletal organization of Chinese hamster ovary and AtT-20 cells expressing membrane PAM. Expression of membrane PAM also alters cytoskeletal organization, demonstrating the presence of endogenous proteins that can mediate this effect. Significant amounts of both PAM and Kalirin fractionate with cytoskeletal elements. Since cytoskeletal organization is critical for exocytosis, constitutive-like and regulated secretions were evaluated. Whereas the constitutive-like secretion of adrenocorticotropic hormone (ACTH) is increased by expression of membrane PAM, regulated secretion is eliminated. Expression of Kalirin in AtT-20 cells expressing membrane PAM restores stimulated secretion of ACTH. Thus, Kalirin or its homologue may be essential for regulated secretion, and the PAM-Kalirin interaction may coordinate intragranular with cytosolic events.

Kalirin, a cytosolic protein with spectrin-like and GDP/GTP exchange factor-like domains that interacts with peptidylglycine alpha-amidating monooxygenase, an integral membrane peptide-processing enzyme.

Although the integral membrane proteins that catalyze steps in the biosynthesis of neuroendocrine peptides are known to contain routing information in their cytosolic domains, the proteins recognizing this routing information are not known. Using the yeast two-hybrid system, we previously identified P-CIP10 as a protein interacting with the cytosolic routing determinants of peptidylglycine alpha-amidating monooxygenase (PAM). P-CIP10 is a 217-kDa cytosolic protein with nine spectrin-like repeats and adjacent Dbl homology and pleckstrin homology domains typical of GDP/GTP exchange factors. In the adult rat, expression of P-CIP10 is most prevalent in the brain. Corticotrope tumor cells stably expressing P-CIP10 and PAM produce longer and more highly branched neuritic processes than nontransfected cells or cells expressing only PAM. The turnover of newly synthesized PAM is accelerated in cells co-expressing P-CIP10. P-CIP10 binds to selected members of the Rho subfamily of small GTP binding proteins (Rac1, but not RhoA or Cdc42). P-CIP10 (kalirin), a member of the Dbl family of proteins, may serve as part of a signal transduction system linking the catalytic domains of PAM in the lumen of the secretory pathway to cytosolic factors regulating the cytoskeleton and signal transduction pathways.

Identification of the promoter for the gene encoding the bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase.

The gene encoding rat peptidylglycine alpha-amidating monooxygenase (PAM) contains 26 protein-coding exons. We identified two non-overlapping genomic clones encoding the 5' untranslated region (UTR) of the PAM gene. Exon 1 has 69 nucleotides flanked by perfect splice acceptor and donor sites, with a TATA motif 25 nucleotides upstream. Exon 0 lacks TATA or CAAT motifs and is embedded in a G + C-rich 800-nucleotide CpG island. The major products identified by RNase protection initiated in exon 0; only a minority of mRNAs initiated in exon 1. 5'-rapid amplification of cDNA ends (RACE) identified the same major transcriptional start sites in exon 0 in the atrium and neurointermediate pituitary. The 2.0-kb fragment upstream of exon 0 and the 1.3-kb fragment upstream of exon 1 were placed upstream of a luciferase-based reporter gene in both sense and antisense orientations. Expression of luciferase was observed in neuroendocrine and nonneuroendocrine cells with both sense constructs. A 0.2-kb fragment of the exon 0 PAM promoter containing multiple GC box elements supported expression of luciferase activity in all cell types. Expression of reporter genes in cells that do not normally express PAM suggests a need for more upstream or intronic information, a role for methylation, or a need for chromatin scaffolding for tissue-specific expression of the endogenous gene.

Dopaminergic regulation of secretory granule-associated proteins in rat intermediate pituitary.

The biosynthesis of peptides requires the synthesis of the prohormone, several biosynthetic processing enzymes, and other granule constituents. We have investigated the regulated expression of proopiomelanocortin (POMC) and five enzymes essential for the processing of POMC to smaller, bioactive peptides in intermediate pituitary melanotropes. Rats were treated with a dopaminergic agonist (bromocriptine) or antagonist (haloperidol) for periods ranging from 1 h to 5 days, followed by analyses of mRNA levels and protein biosynthetic rates. Multiplex RNase protection assays showed that bromocriptine treatment caused a striking decrease in POMC mRNA levels, and significant decreases in mRNA levels for prohormone convertase 2 (PC2), carboxypeptidase H (CPH), and peptidylglycine alpha-amidating monooxygenase (PAM). Smaller increases in mRNA levels were seen after haloperidol stimulation. Protein biosynthetic rates changed more profoundly than mRNA levels at short drug treatment times, indicating a role for translational effects after treatment with bromocriptine and with haloperidol. The homogeneous population of melanotropes in the intermediate lobe of the pituitary allows a quantitative analysis of transcript levels and biosynthetic rates. POMC mRNA levels are 200-1,000-fold higher than levels of any of the processing enzyme mRNAs, and POMC biosynthetic rates exceed those of PC2, PC1, and PAM by 1,000-10,000-fold.

PACE4: a subtilisin-like endoprotease prevalent in the anterior pituitary and regulated by thyroid status.

Low stringency screening of a rat hypothalamic complementary DNA library for additional members of the subtilisin-like prohormone convertase (PC) family identified rat PACE4, which is 90% identical to human PACE4 in amino acid sequence, with much lower similarity to rat PC1, PC2, furin, PC4, or PC6. The rat PACE4 sequence has the Asp-His-Ser catalytic site triad, an Arg-Gly-Asp potential integrin binding site, and three potential sites for N-linked glycosylation. Rat PACE4 has a long COOH-terminal region, which is very rich in Cys residues (15%). The unique signal sequence of rat PACE4 mediates translocation across microsomal membranes during in vitro translation and secretion of PACE4 from stably transfected fibroblast cells. Rat PACE4 has a tissue and cell line distribution unlike any reported PC, including human PACE4, with high expression in the anterior pituitary and readily detectable expression in several brain regions, the atrium, and the ventricle; negligible PACE4 messenger RNA (mRNA) is detected in neurointermediate pituitary and many nonneuroendocrine tissues. PACE4 mRNA is prevalent in Buffalo rat liver and GH3 cells and present at low levels in AtT-20 cells, whereas it is undetectable in several other cell lines. In situ hybridization coupled with immunocytochemistry revealed that PACE4 is produced by somatotropes, mammotropes, and corticotropes, whereas less PACE4 mRNA was detected in thyrotropes. PACE4 mRNA levels in anterior pituitary are strikingly regulated by thyroid status, with more than a 10-fold increase seen from hypothyroid to hyperthyroid animals. The prevalence of PACE4 in anterior pituitary and the striking effect of thyroid status on PACE4 expression suggest a specific role for PACE4 in processing neuroendocrine peptides.