Search

Polarized epithelial cells secrete specific proteins through their apical or basolateral membrane. In the present study, we have expressed the human prosomatostatin cDNA in the pig kidney epithelial cell line (LLC-PK1) and monitored the processing and release of the somatostatin-related peptides. Analysis by high-performance liquid chromatography and radioimmunoassay of the somatostatin-related peptides synthesized by the transfected cells showed that the LLC-PK1 cells released prosomatostatin and somatostatin-28 (S-28) in the culture medium. Furthermore, when the cells were polarized, we observed release of prosomatostatin from both membrane domains (apical and basolateral), while liberation of S-28 was mostly from the basolateral side. This observation suggests that, in these cells, the proprotein convertase(s) responsible for prosomatostatin processing is(are) associated with the basolateral secretory pathway.

Detailed studies on the effects of the ionophore monensin upon synthesis, maturation, and intracellular transport of pro-opiomelanocortin in cultures of rat pituitary intermediate lobe cells have been carried out. When added at concentrations larger than 5 X 10(-8) M monensin significantly inhibited protein synthesis by cultured intermediate lobe cells. Pro-opiomelanocortin synthesis was also reduced proportionally to the overall rate of protein synthesis. During pulse-chase experiments, monensin when added at a concentration of 10(-5) M at the beginning of the chase incubation completely inhibited the proteolytic processing of pro-opiomelanocortin. Using a subcellular fractionation procedure of intermediate lobe cell extracts on Percoll gradients, we were able to show that after the addition of monensin (10(-5) M), labeled pro-opiomelanocortin molecules synthesized during a 15-min pulse-incubation were recovered intact after a 2-h chase, in the fractions of the density gradient corresponding to the rough endoplasmic reticulum and Golgi elements. No maturation products or precursor molecules entered the granule fractions as observed in nontreated cells. Taken together these results strongly suggest that monensin blocks the intracellular transport of newly synthesized pro-opiomelanocortin molecules at the Golgi level and that inhibition of proteolytic processing is due to the failure of the prohormone to enter the cell compartment (probably the secretion granules) where maturation proteases are located.

Pro-opiomelanocortin, the common glycoprotein precursor to adrenocorticotropin and beta-lipotropin, is the most abundant protein synthesized in rat neurointermediate lobes. Dissected rat neurointermediate lobes were incubated in the presence of canavanine, an analog of arginine, to determine (a) whether canavanine could be incorporated into pro-opiomelanocortin molecules and (b) if incorporation occurs, whether there is any effect on the processing mechanism of the prohormone. Preincubation of rat neurointermediate lobes for 16 h in the presence of 10 mM canavanine results in the production of pro-opiomelanocortin molecules in which most, if not all, the arginine residues have been replaced by canavanine. Identification of canavanine-containing pro-opiomelanocortin forms was done by two-dimensional electrophoresis, tryptic and chymotryptic peptide mapping, as well as by analysis, on polyacrylamide gels in the presence of sodium dodecyl sulfate, of the fragments resulting from a partial digestion with chymotrypsin. During pulse-chase experiments, canavanine-containing pro-opiomelanocortin molecules were found to be processed at a much slower rate than the normal precursor forms: after a 2-h chase, conversion of approximately 25% of the analog-containing prophormone was observed compared to 83% of the nonanalog-containing precursors. Moreover, the small proportion of canavanine-containing precursor molecules which had undergone cleavage during the chase yielded atypical large molecular weight peptides. These results indicate that canavanine incorporation into neurointermediate lobe proteins considerably slows down the conversion of pro-opiomelanocortin into its different end products.

Pro-opiomelanocortin has a signal peptide of 26 amino acids which is very hydrophobic like the other signal peptides. The large precursor has two forms which differ in their carbohydrate content. The presence of pairs of amino acids at the proposed sites of cleavage to release ACTH, beta-MSH and endorphin is a feature described in 1967 for beta-MSH and the C-terminal fragment which is now known as beta-endorphin. It is proposed that other neuropeptides will be biosynthesized through a similar type of processing.

Pulse and pulse-chase studies of the peptide hormones synthesized in the rat pars intermedia showed that during the maturation of pro-opiomelanocortin (the adrenocorticotropin-beta-lipotropin (ACTH-beta-LPH) precursor) the N-terminal portion of the precursor was released as a glycopeptide of relative mass of 17 000 - 19 000 and did not undergo further proteolytic cleavage. This peptide is therefore proposed to be the biologically active species related to the amino terminal fragment of pro-opiomelanocortin in the rat pars intermedia.

When isolated rat pars intermedia cells were incubated for 10 min with radioactive amino acids, one major labeled protein with a molecular weight of 30,000 +/- 1500 was extracted. This protein was shown to contain in its sequence the antigenic determinants for corticotropin and beta-melanotropin by immunoprecipitation. When the radioactivity incorporated into this large molecular weight protein during the first 10 min was chased by a further incubation in presence of an excess of unlabeled amino acid, the initial protein was degraded into several smaller peptides including beta-endorphin and beta-lipotropin. Another 18,000-dalton peptide was also observed and was tentatively identified as a large molecular form of corticotropin. From the kinetics of the maturation of the initial precursor, it is concluded that the initial cleavage of the 30,000-dalton peptide gives rise to beta-lipotropin and the 18,000-dalton form of corticotropin. beta-Lipotropin is subsequently cleaved to form beta-endorphin.

In the pars intermedia of rat pituitary glands, two forms of a common precursor for corticotropin (ACTH) and beta-lipotropin with apparent molecular weights of 34,000 and 36,000 were resolved by sodium dodecyl sulfate/acrylamide gradient slab gel electrophoresis. High-performance liquid chromatographic analysis of [35S]methionine-labeled tryptic fragments of the two forms of the precursor revealed that both contained copies of ACTH-(1-8) and beta-lipotropin-(61-69) sequences. When biosynthetic studies were performed in the presence of tunicamycin, the 34,000- and 36,000-dalton forms were replaced by a peptide with an apparent molecular weight of 32,000. It was therefore concluded that the 34,000- and 36,000-dalton forms of the precursor represent two glycoprotein variants of similar polypeptides, differing in the number of asparagine-linked carbohydrate moieties. During pulse-chase incubations with [35S]methionine, the precursor forms were cleaved into two major groups of labeled products: (i) beta-endorphin and (ii) a mixture of ACTH fragments closely related to alpha-melanotropin. No ACTH-(1-39) was found at the end of a 2-hr chase period, suggesting that ACTH is not a significant hormone product of the rat pars intermedia.

Studies on the biosynthesis of beta-LPH on the one hand, and of ACTH on the other, have produced a new concept, that of a single precursor form which contains three active molecules. Thus, it is proper to name such a precursor 'pro-opio-melanocortin.' The concept that beta-LPH was a precursor molecule was first put forward in 1967 and was based on both structural forms and biological activities. The discovery that morphine-like substances are part of the C-terminal fragment of beta-LPH brought an additional important biological side product. That, together with the recent demonstration of ACTH as part of a still larger precursor, constitutes an exciting model for the study of peptide hormone biosynthesis. We have shown unambiguously that beta-endorphin is the result of a maturation process from the large precursor, while beta-LPH is an important and transient intermediary. Since it is also present in the brain, our recent results using pars intermedia cells can be applied to study the fabrication and degradation of these molecules in the brain. We expect to see it established that all other neuropeptides are also biosynthesized as larger precursor molecules whose structure at the site of cleavage could well be constituted of two basic amino acids like in the pro-opio-melanocortin.

Proopiomelanocortin, the common glycoprotein precursor to adrenocorticotropin (ACTH) and beta-lipotropin (beta-LPH), is the most abundant protein synthesized in rat neurointermediate lobes. It represents 30% of the total amount of radioactive proteins obtained after a 1-h pulse incubation with [3H]phenylalanine. Several forms of this protein can be separated by a high-resolution two-dimensional gel electrophoresis technique. The three most abundant species which can be reproducibly characterized by their apparent molecular weights (Mr) and isoelectric points (pI) were called form I (Mr 34 000; pI 8.2), form II (Mr 36 000; pI 8.2), and form III (Mr 35 000; pI 7.3). Additional minor forms, representing together approximately 30% of the total forms I, II, and III combined, are also observed. They have very close molecular weights but differ by their isoelectric points. When glycosylation is prevented by tunicamycin, forms I and II are replaced by a new molecule with the same pI of 8.2 but a slightly lower Mr (32 000). This form is referred to as form T1. Similarly, form III is replaced by form T2 (Mr 33 000; pI 7.3). Forms T1 and T2 are supposed to be nonglycoslyated peptides. They were further characterized by microsequencing and peptide mapping. They both have the same N-terminal amino acid sequence with leucine residues in positions 3 and 11, and they both contain identical [3H]phenylalanine-labeled tryptic fragments, two of them corresponding to the sequences 1-8 of ACTH and 61-69 of beta-LPH. However, a limited digestion with the Staphylococcus aureus (V8 strain) protease generates a collection of peptides different for each form. These results suggest the presence of at least two different gene products corresponding to the major forms of proopiomelanocortin in the rat pars intermedia.

Although more and more is known about the nature of neuropeptides, there are two basic questions which need to be looked at in order for the neurophysiologists and the neuropharmacologists to have all the tools necessary for their studies. The first question is the site of biosynthesis of neuropeptides and the second is their biosynthetic pathway. We believe and propose that all neuropeptides will be formed from a larger molecular weight precursor similar to beta-LPH for beta-endorphin. All models of peptide hormones (pro-insulin, pro-PTH, pro-glucagon, beta-lipotropin etc.,) show the same characteristic preferential site of cleavage made of a double basic residue. This is also the type of cleavage recently described by Nakanishi et al. (1979). Using the rat pars intermedia, we showed that this tissue preferentially makes alpha-MSH and beta-endorphin from a large precursor, while beta-LPH and ACTH are short lived intermediates. All products of maturation were identified by chemical analyses leaving no doubt about their nature and identity. We strongly believe that the brain will biosynthesize beta-endorphin and possibly alpha-MSH in similar fashion. We also predict that our results will apply to the other neuropeptides which wil be formed from large precursors. These studies could be very useful in the interpretation of the intricate relationship between neuropeptides and behavioral effects.