Access of a membrane protein to secretory granules is facilitated by phosphorylation.

Peptidylglycine alpha-amidating monooxygenase (PAM), an integral membrane protein essential for the biosynthesis of amidated peptides, was used to assess the role of cytosolic acidic clusters in trafficking to regulated secretory granules. Casein kinase II phosphorylates Ser(949) and Thr(946) of PAM, generating a short, cytosolic acidic cluster. P-CIP2, a protein kinase identified by its ability to interact with several juxtamembrane determinants in the PAM cytosolic domain, also phosphorylates Ser(949). Antibody specific for phospho-Ser(949)-PAM-CD demonstrates that a small fraction of the PAM-1 localized to the perinuclear region bears this modification. Pituitary cell lines expressing PAM-1 mutants that mimic (TS/DD) or prevent (TS/AA) phosphorylation at these sites were studied. PAM-1 TS/AA yields a lumenal monooxygenase domain that enters secretory granules inefficiently and is rapidly degraded. In contrast, PAM-1 TS/DD is routed to regulated secretory granules more efficiently than wild-type PAM-1 and monooxygenase release is more responsive to secretagogue. Furthermore, this acidic cluster affects exit of internalized PAM-antibody complexes from late endosomes; internalized PAM-1 TS/DD accumulates in a late endocytic compartment instead of the trans-Golgi network. The increased ability of solubilized PAM-1 TS/DD to aggregate at neutral pH may play an important role in its altered trafficking.

Dopamine beta-monooxygenase signal/anchor sequence alters trafficking of peptidylglycine alpha-hydroxylating monooxygenase.

Dopamine beta-monooxygenase (DBM) and peptidylglycine alpha-hydroxylating monooxygenase (PHM) are essential for the biosynthesis of catecholamines and amidated peptides, respectively. The enzymes share a conserved catalytic core. We studied the role of the DBM signal sequence by appending it to soluble PHM (PHMs) and expressing the DBMsignal/PHMs chimera in AtT-20 and Chinese hamster ovary cells. PHMs produced as part of DBMsignal/PHMs was active. In vitro translated and cellular DBMsignal/PHMs had similar masses, indicating that the DBM signal was not removed. DBMsignal/PHMs was membrane-associated and had the properties of an intrinsic membrane protein. After in vitro translation in the presence of microsomal membranes, trypsin treatment removed 2 kDa from DBMsignal/PHMs while PHMs was entirely protected. In addition, a Cys residue in DBMsignal/PHMs was accessible to Cys-directed biotinylation. Thus the chimera adopts the topology of a type II membrane protein. Pulse-chase experiments indicate that DBMsignal/PHMs turns over rapidly after exiting the trans-Golgi network. Although PHMs is efficiently localized to secretory granules, DBMsignal/PHMs is largely localized to the endoplasmic reticulum in AtT-20 cells. On the basis of stimulated secretion, the small amount of PHMs generated is stored in secretory granules. In contrast, the expression of DBMsignal/PHMs in PC12 cells yields protein that is localized to secretory granules.

Signaling mediated by the cytosolic domain of peptidylglycine alpha-amidating monooxygenase.

The luminal domains of membrane peptidylglycine alpha-amidating monooxygenase (PAM) are essential for peptide alpha-amidation, and the cytosolic domain (CD) is essential for trafficking. Overexpression of membrane PAM in corticotrope tumor cells reorganizes the actin cytoskeleton, shifts endogenous adrenocorticotropic hormone (ACTH) from mature granules localized at the tips of processes to the TGN region, and blocks regulated secretion. PAM-CD interactor proteins include a protein kinase that phosphorylates PAM (P-CIP2) and Kalirin, a Rho family GDP/GTP exchange factor. We engineered a PAM protein unable to interact with either P-CIP2 or Kalirin (PAM-1/K919R), along with PAM proteins able to interact with Kalirin but not with P-CIP2. AtT-20 cells expressing PAM-1/K919R produce fully active membrane enzyme but still exhibit regulated secretion, with ACTH-containing granules localized to process tips. Immunoelectron microscopy demonstrates accumulation of PAM and ACTH in tubular structures at the trans side of the Golgi in AtT-20 cells expressing PAM-1 but not in AtT-20 cells expressing PAM-1/K919R. The ability of PAM to interact with P-CIP2 is critical to its ability to block exit from the Golgi and affect regulated secretion. Consistent with this, mutation of its P-CIP2 phosphorylation site alters the ability of PAM to affect regulated secretion.

Phosphorylation of cytosolic domain Ser(937) affects both biosynthetic and endocytic trafficking of peptidylglycine alpha-amidating monooxygenase.

Peptidylglycine alpha-amidating monooxygenase (PAM), a bifunctional enzyme, catalyzes the COOH-terminal amidation of bioactive peptides. In test tube assays, PAM is phosphorylated by protein kinase C at Ser(937). The roles of phosphorylation and dephosphorylation of Ser(937) in the biosynthetic and endocytic trafficking of integral membrane PAM were examined using an antiserum specific for the phosphorylation of Ser(937) and using AtT-20 cells expressing membrane PAM in which Ser(937) was mutated to Ala or Asp. Although phosphorylation at Ser(937) can occur while PAM is in the endoplasmic reticulum, early steps in the biosynthetic trafficking of membrane PAM were not affected by Ser(937) phosphorylation. The inability to phosphorylate PAM/S937A increased its intracellular degradation and decreased secretion of the soluble monooxygenase portion of PAM. In contrast, the biosynthetic trafficking of PAM/S937D was indistinguishable from wild-type PAM. Despite the fact that Ser(937) is adjacent to the only Tyr-based internalization motif in PAM, internalization and trafficking through early endosomes were unaffected by phosphorylation. However, PAM antibody internalized by wild-type PAM acquired a perinuclear localization, while antibody internalized by PAM/S937A was routed to lysosomes, and antibody bound to PAM/S937D maintained a dispersed, punctate pattern. In cells stimulated with phorbol ester, phosphorylation of Ser(937) increased and phosphorylated PAM accumulated in large vesicular structures. Therefore, phosphorylation of PAM-1 at Ser(937) directs newly synthesized and internalized protein away from lysosomes, while dephosphorylation is needed for a different step in the late endocytic pathway.

POMC-related products in the intermediate pituitary of the amphibian, Bufo marinus: differential subcellular processing in the Golgi and secretory granules.

In the intermediate pituitary of the anuran amphibian, Bufo marinus, the N-acetylation of ACTH(1-13)-NH2 to yield alpha-MSH occurs as a cosecretory processing event, whereas the N-acetylation of beta-endorphin occurs as a posttranslational processing event. To understand how these two N-acetylation reactions are segregated, B. marinus intermediate pituitary cells were analyzed by immunogold labeling electron microscopy, and by using an ultracentrifugation procedure. The immunogold labeling studies indicated that ACTH(1-13)-NH2-related immunoreactivity was colocalized with N-acetylated beta-endorphin-related immunoreactivity in secretory granules. Furthermore, ACTH(1-13)-NH2-related immunoreactivity was not detected in either the ER or the Golgi. N-Acetylated beta-endorphin-related immunoreactivity, however, was detected in the Golgi. Ultracentrifugation analysis revealed that in an ER/microsomal fraction, beta-LPH-sized and nonacetylated beta-endorphin-sized immunoreactive material were present in a molar ratio of 1:2. No N-acetylated forms of beta-endorphin were detected in the ER/microsomal fraction. In a Golgi/secretory granule fraction, the molar ratio of beta-LPH to beta-endorphin was 1:9 with 58% of the beta-endorphin being N-acetylated. Collectively, these data support the following hypotheses. The proteolytic cleavage of ACTH (1-39) to yield ACTH (1-13)-NH2 is a late processing event occurring in secretory granules. The cleavage of beta-LPH to yield nonacetylated beta-endorphin is an early processing event that may occur in the ER or the Golgi. Because N-acetylated beta-endorphin and nonacetylated ACTH(1-13)-NH2 are colocalized in secretory granules, it appears, therefore, that the N-acetylation of beta-endorphin is completed prior to loading into secretory granules. Thus, there is a spatial and temporal separation of the posttranslational processing events associated with the beta-LPH portion and ACTH portion of the POMC biosynthetic pathway in amphibian intermediate pituitary cells.

Melanotropes of the lizard, Anolis carolinensis, lack N-acetylating mechanisms for both alpha-melanocyte-stimulating hormone and beta-endorphin.

In order to determine whether Anolis carolinensis intermediate pituitary cells have the capacity to N-acetylate either ACTH(1-13)NH2 or beta-endorphin during secretion, individual intermediate pituitary explants were incubated in DMEM/CO2 for 24 h at 28 degrees C. Although alpha-melanocyte-stimulating hormone (alpha-MSH)- and beta-endorphin-related products were spontaneously released into the medium, none of these forms were N-acetylated. It appears that unlike most gnathostomes, A. carolinensis has secondarily lost the POMC-specific N-acetylation mechanisms. A ramification of this observation is that the alpha-MSH for A. carolinensis is ACTH(1-13)NH2.

The posttranslational modification of beta-endorphin in the intermediate pituitary of the toad, Bufo marinus, includes processing at a monobasic cleavage site.

Fractionation of an acid extract of 15 B. marinus intermediate pituitaries by a combination of gel filtration chromatography and cation exchange chromatography revealed one major and five minor forms of beta-endorphin in this tissue. Based on reversed-phase HPLC and immunological properties, as well as amino acid composition and primary sequence analysis, it was deduced that the sequence of the major form of B. marinus beta-endorphin is N-acetyl-YGGFMTPE. Overall, the steady-state analyses of the minor forms of beta-endorphin indicated that the posttranslational processing of beta-endorphin in the toad intermediate pituitary includes endoproteolytic cleavage at both paired basic and monobasic cleavage sites.

Detection and partial characterization of proopiomelanocortin-related end-products from the pars intermedia of the toad, Bombina orientalis.

Steady-state analyses were performed on the proopiomelanocortin (POMC)-related end-products present in acid extracts of the pars intermedia of the anuran amphibian, Bombina orientalis. Sephadex G-75 gel filtration chromatography indicated that immunoreactive alpha-MSH-sized material and N-acetylated beta-endorphin-related material are the major POMC-related products present in this tissue. The alpha-MSH-sized immunoreactivity was further fractionated by reversed phase HPLC. The major peak of immunoreactivity isolated by this procedure eluted with the same retention time as synthetic ACTH(1-13)amide. Cation exchange chromatography supported the conclusion that the major storage form of alpha-MSH in the pars intermedia of Bombina is ACTH(1-13)amide. Analysis of Bombina pars intermedia in culture indicated that mono-acetylated and di-acetylated alpha-MSH were the major forms of alpha-MSH secreted into the medium. The major peak of N-acetylated beta-endorphin-related material was further analyzed by cation exchange chromatography and Sephadex G-25 gel filtration column chromatography. The major storage form of beta-endorphin in this tissue is N-acetylated, has a net positive charge at pH 2.75 of +1, and has an apparent molecular weight of 1.2K. The beta-endorphin present in the pars intermedia of this tissue does not undergo further N-acetylation at the time of secretion. These results indicate that in the pars intermedia of the archaeobatrachian, Bombina orientalis, the N-acetylation of alpha-MSH is a cosecretory processing event, whereas N-acetylation of beta-endorphin is a post-translational processing event. These results are compared to other archaeobatrachian and neobatrachian pituitary POMC systems that have been analyzed.

Differential mechanisms for the N-acetylation of alpha-melanocyte-stimulating hormone and beta-endorphin in the intermediate pituitary of the frog, Xenopus laevis.

Immunohistochemical analysis of the pituitary of Xenopus laevis revealed the colocalization of alpha-melanocyte-stimulating-hormone (MSH)-related immunoreactivity and N-acetyl-beta-endorphin-related immunoreactivity in the cells of the intermediate pituitary. In order to determine whether the immunoreactive N-acetylated beta-endorphin is released in parallel with the immunoreactive alpha-MSH, intermediate pituitaries were incubated in L-15 medium for 24 h. The medium and an acid extract of the intermediate pituitaries from each incubation were separately fractioned by a combination of gel filtration chromatography, reverse-phase high-performance liquid chromatography, and cation exchange chromatography. In the intermediate pituitary extract, the major form of alpha-MSH had chromatographic properties which corresponded to nonacetylated alpha-MSH (ACTH)(1-13)amide; whereas the major form of beta-endorphin had an apparent molecular weight of 1.2 kDa and was N-acetylated. The 1.2-kDa form of beta-endorphin and ACTH(1-13)amide were present in equimolar amounts. Analysis of the medium indicated that both end products were released in parallel. However, as reported in the literature, there was a significant increase in the N-acetylation of ACTH(1-13)amide during secretion. There was no further processing of beta-endorphin during secretion. Collectively, these observations indicate that in the intermediate pituitary of X. laevis there are separate mechanisms for the N-acetylation of alpha-MSH and beta-endorphin.

Detection of N-acetylated forms of beta-endorphin and nonacetylated alpha-MSH in the intermediate pituitary of the toad, Bufo marinus.

Steady-state analysis of the acid extracts of the intermediate pituitary of the toad, Bufo marinus, revealed the presence of multiple forms of beta-endorphin and alpha-MSH. Approximately 98% of the immunoreactive beta-endorphin was N-acetylated. The major form of N-acetylated beta-endorphin, which represented 81.5% of the total beta-endorphin recovered from this tissue, had an apparent molecular weight of 1.2 kDa and a net charge of +1 at pH 2.75. Approximately 98% of the immunoreactive alpha-MSH present in the Bufo intermediate pituitary had reverse phase HPLC properties similar to the nonacetylated form of alpha-MSH, ACTH(1-13)amide. These observations are in agreement with studies on the intermediate pituitary of the frog, Xenopus laevis, which have shown that the N-acetylation of alpha-MSH in this species is a cosecretory processing event, whereas the N-acetylation of beta-endorphin is a posttranslational processing event (2, 5, 15). These observations indicate that the N-acetylation of beta-endorphin and alpha-MSH occurs at distinct subcellular sites in intermediate pituitary cells of anuran amphibians. The Bufo intermediate pituitary will serve as a good model system for studying these novel N-acetyltransferase reactions.

The molecular evolution of neuropeptides: prospects for the '90s.

Three distinct opioid precursors have been identified in the central nervous system of mammals: proopiomelanocortin (POMC), proenkephalin, and prodynorphin. These precursors are derived from separate genes, synthesized in distinct neurons, and yield unique sets of opioid end products. This review will discuss the general mechanisms involved in the biosynthesis of neuropeptide precursors and consider the roles of posttranscriptional and posttranslational processing mechanisms in the generation of multiple sets of end products from a single gene. In addition, techniques that can be used for isolating and characterizing neuropeptide genes, mRNAs, and end products will be reviewed. These introductory comments will serve as the framework for a discussion of the phylogeny of the opioid precursors in the major groups of non-mammalian vertebrates.

The isolation of multiple forms of beta-endorphin from the intermediate pituitary of the Australian lungfish, Neoceratodus forsteri.

The pituitary of the Australian lungfish, Neoceratodus forsteri, was screened immunohistochemically with heterologous antisera specific for either the C-terminal of mammalian beta-endorphin or the acetylated N-terminal of beta-endorphin. Immunopositive cells were only detected with the N-terminal specific antiserum; these cells were restricted to the intermediate pituitary. Acid extracts of the intermediate pituitary were fractionated by Sephadex gel filtration chromatography, CM cation exchange chromatography and reverse phase HPLC. Fractions were analyzed by radioimmunoassay (RIA) with a N-acetyl specific beta-endorphin RIA and by radioreceptor assay for the presence of opiate active forms of beta-endorphin. Both immunoreactive and opiate active forms of beta-endorphin were detected. Of the total beta-endorphin-related material isolated from the intermediate pituitary, approximately 97% was detected with the N-terminal specific RIA and approximately 3% was detected by the radioreceptor assay. The N-acetylated immunoreactive beta-endorphin could be separated into two forms. The major form had an apparent molecular weight of 3.2 Kda. This material had a net charge at pH 2.5 of +5. The minor form of immunoreactive beta-endorphin had an apparent molecular weight of 1.4 Kda and a net charge at pH 2.5 of +1. Neither immunoreactive form exhibited receptor binding activity in the radioreceptor assay. A single peak of opiate active beta-endorphin was detected. This material had an apparent molecular weight of 3.5 Kda and a net charge at pH 2.5 of +7.