Los Angeles

Date:
1981
Reference:
PP/CRI/E/1/29/18
Catalogue details

Licence: In copyright

Credit: Los Angeles. Source: Wellcome Collection.

    33/41
    PROOPIOMELANOCORTIN: A MODEL FOR THE REGULATION OF EXPRESSION OF NEUROPEPTIDES IN PITUITARY AND BRAIN Edward Herbert, Neal Birnberg, Jean-Claude Lissitsky, Olivier Civelli, and Michael Uhler Department of Chemistry, University of Oregon, Eugene, Oregon 97403 (A glossary on pages 24-25 defines terms italicized in this text.) A variety of neuroactive peptides which have been discovered in higher animals recently has provided us with a number of very promising candidates for neuro transmitter and neuromodulator molecules in the brain. A partial list of these peptides is presented in Table I. Some of the peptides in Table I have been shown to mediate specific behavioral responses in animals. Hypo thalamic releasing factors, for example, regulate the se- play a role in growth and development and mediate re- ature cycles. The opioid peptides, met- and leu-enkeph- alin, dynorphin and ß-endorphin, among other effects, raise the threshold to pain. Other neuropeptides have been implicated in the induction of sleep and regulation of temperature in mammals (bombesin). Small Neuroactive Peptides from Recombinant DNA technology and other tools of the molecular biologist have revealed that small neuroac tive peptides such as the enkephalins, endorphins, so matostatin, gastrin, and ACTH are derived from pro- neuroactive peptides (Figure 1). Why has nature made such large precursors for the production of such small peptides? First, neuropeptides must be cut from precur sor proteins by proteolytic enzymes and undergo other chemical modifications to be activated and secreted. Chemical modifications like glycosylation (addition of carbohydrate side chains), phosphorylation, acetyla- tion, and methylation occur in a well-defined order as newly synthesized proteins move through the endo plasmic reticulum, Golgi complex, and other compart ments of the cell on their way to being secreted. The in formation that specifies the sites of modification and hydrolytic cleavages is encoded in the amino acid se quences of secretory proteins. For example, the interac tion of a secretory protein with the membranes of the endoplasmic reticulum is specified by a sequence of 15 to 30 amino acids called a signal sequence (Blobel and Dobberstein, 1975). This sequence is almost always at the amino terminus of the secretory protein and is removed by a specific proteolytic enzyme in the mem branes of the endoplasmic reticulum as the nascent pep tide chain attached to the ribosome penetrates the membrane during biosynthesis (Jackson and Blobel, 1977). Glycosylation of the protein also requires a spe- However, not all such sites in a protein are glycosy lated; therefore, other features of the protein (confor mation, for example) may also play a role in specifying glycosylation. Chemical modifications after synthesis, such as acetylation, methylation, or cleavage, may acti- age site. Thus, a portion of the sequence of the precur sor protein is likely to be required for the interactions involved in the compartmentalization of proteins and A second reason for large precursors is that they may contain the sequences of other biologically active pep tides. Thus, the precursor of ACTH is also the precur sor of ß-endorphin and other hormones (Mains et al., 1977; Roberts and Herbert, 1977a, b). The precursor for met-enkephalin contains the sequence of leu-enkephalin (Kimura et al., 1980), and oxytocin and vasopressin share the same precursor with the neurophysins (bind ing proteins for oxytocin and vasopressin [Brownstein et al., 1980; see Figure 1]). This is reminiscent of the large viral polyproteins that contain several entities such as proteolytic enzymes and maturation factors that become active only after they are cut out of the parent protein during maturation of the virus (Korant, 1981). By analogy, it seems appropriate to refer to precursors of ACTH, enkephalins, oxytocin, and vasopressin as cellular polyproteins. Since the precursor to ACTH and jßrendorphin is the best characterized of the neural poly proteins, we will consider it in more detail. This pre- cyte-stimulating hormones (MSH), ACTH, and ß- endorphin, and is called pro-opiomelanocortin (POMC). Structure and Processing of Pro-opiomelanocortin Nakanishi and co-workers (1979) have recently de termined the complete amino acid sequence of bovine POMC by recombinant DNA technology. A partial se quence of POMC from mouse pituitary cells has also TABLE I Partial list of neurotransmitter and neuromodulator candidates Pituitary Peptides* Adrenocorticotropin (ACTH) /3-endorphin a-melanocyte-stimulating hormone Oxytocin Vasopressin Neurophysins I and II Hypothalamic Releasing Hormones* Peptides from Brain and Intestine* Vasoactive intestinal peptide Cholecystokinin Substance P Neurotensin Enkephalins Dynorphin Gastrin Some of these peptides are also found at other sites in the animal. been determined by Roberts et al. (1979) using the same methods. An interesting feature of POMC molecules (Figure 2) is that pairs of lysine and arginine residues flank each of the biologically active domains of the molecule (ACTH, ^-endorphin, N-terminal portion, etc.). Since trypsin requires basic amino acids for its ac tion, a trypsin-like enzyme or enzymes may be involved in the release of hormones from the precursor. This is similar to the conversion of pro-insulin to insulin des cribed by Steiner et al. (1967). Also, the MSH sequence is repeated three times in the molecule (in the N-termi nal region [7 -MSH], ACTH region [a-MSH], and ß- LPH region [0-MSH]). All of these sequences are highly conserved from one species to another, suggest ing that they have an important function. Another im portant finding is that the POMC molecule is present in both the anterior and intermediate lobes of the pituitary and possibly in a number of extrapituitary sites, includ ing the hypothalamus and amygdala. The presence of POMC in various tissues raises intriguing questions, both because the molecule is processed to different hormones in these sites and because the release of ACTH/endorphin peptides is regulated differently in these sites. Processing of POMC in Mouse Pituitary Tumor Cells (AtT-20-D, 6v Cells) The most complete studies of processing of POMC have been carried out with a mouse pituitary tumor cell line (AtT-20-D l6 , line) which secretes ACTH and ß- LPH and mimics the functions of the normal anterior pituitary cells that produce ACTH and endorphins (corticotrophs) (Herbert et al., 1980; Eipper and Mains, 1980; Roberts et al., 1978). AtT-20-D, 6v cells were incu bated with radioactive amino acids and/or sugars, and the fragments produced from each domain of the mole cule during processing were isolated by immunoprecipi- tation with antibodies specific for ACTH, endorphin, or the N-terminal region of the molecule. The immunopre- cipitation procedure succeeded because each of the an tibodies reacted with its antigenic determinant whether it was present in the large molecular weight precursor, intermediate peptide, or end product. The immunopre- large precursors for small peptide hormones ¡of precursor peptide ami^o^ids AVP Neurophysin *AVP - arginine vasopressin II neuropeptides are derived from large p