Alex N. Eberle

Irreversible stimulation of Xenopus melanophores by photoaffinity labelling with p‐azidophenylalanine13‐α‐melanotropin

α-melanotropin induces pigment dispersion in nmelanophores and pigment formation in melanocytes nand melanoma cells. The pigmentdispersingeffect nof MSH is completely reversible in vitro, as the nmelanosomes reaggregate readily upon removal of nthe hormone. The primary site of action of α-MSH nappears to be a cell surface receptor, which can be nstimulated by two different portions of the α-MSH nmolecule, namely the central (-Glu-His-Phe-Arg- nTrp-) and the C-terminal (-Gly-Lys-Pro-Val . nNH₂) message sequences. The C-terminal residue nis essential for the interaction with the receptor, since nits absence reduces the biological activity considerably n(in preparation); the structure of its side-chain, however, nis less important, and it may therefore contain nan affinity label.

Calcium sites in MSH stimulation of xenopus melanophores: Studies with photoreactive α-MSH

Abstract Photo-affinity labelling of MSH receptors on tail-fin melanophores of Xenopus tadpoles with p -azidophenylalanine 13 -α-MSH ([Pap 13 ]-α-MSH) or p -azidophenylacetyl-serine 1 -α-MSH ([Apac-Ser 1 ]-α-MSH) results in a long-lasting stimulation of the melanophores which cannot be reversed despite continuous washing. The generation of this irreversible response is inhibited when photo-affinity labelling is performed in a Ca 2+ -free medium or in the presence of Ca 2+ antagonists. The irreversible stimulation produced in normal medium is completely reversed upon removal of Ca 2+ , but is not affected by Ca 2+ antagonists or melatonin. Re-addition of Ca 2+ after temporary removal restores the irreversible stimulation, even in the presence of Ca 2+ antagonists or melatonin. This proves that covalent α-MSH-receptor complexes remain fully functional despite temporary deprivation of Ca 2+ . Racemized α-MSH, which binds ‘tightly’ to the receptor and produces a long-lasting effect, is removed from the receptor in Ca 2+ -free medium, but not by Ca 2+ antagonists or melatonin. These results confirm earlier results showing that at least 2 Ca 2+ sites are involved in α-MSH action, one associated with MSH-receptor binding and the other with the subsequent generation of the effect. The dual role of Ca 2+ is not the result of EGTA present; it is specific (Mg 2+ has no effect) and is identical for the two different photoreactive α-MSH derivatives.

Photoaffinity labelling of MSH receptors reveals a dual role of calcium in melanophore stimulation

UV irradiation of Xenopus laevis melanophores in the presence of photoreactive [Pap13]-ol-MSH induces irreversible pigment dispersion [ 13. It appears that specific covalent labelling of MSH receptors results in the generation of a continuous signallasting for several hours. This longlasting effect makes it possible to study the involvement of various factors, such as Ca*“, at the different steps (see [2]) of hormonal signal transmission. Ca*+ is well known to be indispensable for mediating the effect of (u-MSH on melanophores of several species [3,4]. From earlier studies with melanophores of Rana pipiens [S] and Xenopus laevis [6] it was concluded that the interaction of c+MSH with its receptor leads to the activation of an adenylate cyclase system, as was shown with mouse melanoma cells [ 71. The subsequent pigment dispersion is controlled by cAMPand does not depend on extracellular Ca*+, since the dispersion can be brought about by dbcAMP in the absence of Ca*+ [3,8]. Thus the effect of Ca*” seems to precede the action of CAMP; however, the precise site of Ca*+ action remained unclear. Photoaffmity labelling of MSH receptors enabled us to distinguish between receptor-associated and post-receptor Ca*+ requirement. Here we show that Ca*+ is essential for hormone-receptor binding, for coupling of the hormone receptor to adenylate cyclase and/or for activation of the enzyme.

Hormone—receptor interactions: Melanotropic activities of covalent serum albumin complexes with α-melanotropin, α-melanotropin fragments, and enkephalin

The investigation of the biological properties of about 60 cu-melanotropin analogues and fragments has led to the conclusion that this tridecapeptide consists of two message sequences which are capable of triggering the hormonal stimulus independently of one another [l-3] . The first message sequence is centered around -His-Phe-Arg-Trp[4] and the second within the C-terminal tetrapeptide, -Gly-Lys-ProVal.NH2 [l] . The N-terminal tetrapeptide, Ac-SerTyr-Ser-Met.OH, is devoid of melanotropic activity; it potentiates, however, the activity of the C-terminal nonapeptide about loo-fold [2] : A recent claim by Medzihradszky and Medzihradszky-Schweiger [S] that the N-terminal tetrapeptide,‘as well as a number of peptides related to the enkephalins, exhibit tielanosome-dispersing properties necessitated a re-investigation of our results. At the same time the question should be answered whether the fragments of ru-MSH elicit their stimulus by the same mechanism as the parent hormone, namely by acting on a cell surface membrane receptor [6] . To this end, (u-MSH derivatives and fragments have been attached covalently to human serum albumin, and the complexes have been purified, analyzed and tested for melanotropic activity in

α-Melanotropin Receptors: Non-Identical Hormonal Message Sequences (Active Sites) Triggering Receptors in Melanocytes, Adipoctytes and CNS Cells

α-Melanotropin (α-melanocyte-stimulating hormone, α-MSH), a pituitary tridecapeptide, exhibits strong effects on a variety of tissues. The best known is certainly the dramatic impact on the color change of cold-blooded vertebrates where α-MSH causes darkening of the skin by dispersion of the melanophore pigment granules. a-MSH seems also to control—at least partially—the pigmentation of mammals and men (35). Only a small number of extrapigmentary effects of α-melanotropin will be mentioned here as e.g. the lipolytic activity in rabbit adipose tissue (7, 45), the effects on learning and the central nervous system (11, 13, 46, 47, 58), and the activity on sebum secretion in the rat (49, 60).

AGE-DEPENDANT VARIATION IN FUNCTION OF THE ADRENAL CORTEX IN RATS

Proopiomelanocortins (POMC) secreted concomitantly with ACTH may be involved in control of adrenal growth and function with changing needs for POMC peptides during development. This might explain an increased ACTH response to stress, which was found in 2-4 week old, compared to adult rats. Therefore, the sensitivity of the adrenal to ACTH was compared between prepubertal (pp)(25 days) and young adult (ya)(70 days) rats. In vivo: In dexamethasone-blocked rats, corticosterone (c) 20 min after 25 ng ACTH1-24/100 g bw i.v. was significantly higher (50.3±2.1 ng%) in pp compared to ya rats (41.0±1.8 ng%). In contrast, 5 ng ACTH/100 g bw could not stimulate c in pp (4.8±1.6 ng%), but markedly in ya (30.8±1.7 ng%). For a comparable c-response in pp rats, 10 ng of ACTH/100 g bw was reguired. In vitro, cells prepared by collagenase dispersion from inner zones of the adrenal cortex, revealed similar results: 1 pg of ACTH elicits a significant response in ya, but 5 pg are needed in pp rats. ED50 was correspondingly increased from 2×10−12 to 1×10−11 M. Maximal responses were similar and dose response curve parallel. This suggests a protective mechanism against an over-production of c, due to large amounts of ACTH released with other POMC-peptides needed for adrenal growth in prepubertal animals.

α-MSH induces changes in protein phosphorylation in Cloudman S-91 mouse melanoma cells

α-MSH stimulates pigment synthesis in melanocytes and melanoma cells by interacting with a specific membrane receptor/adenylate cyclase system which activates tyrosinase, the key enzyme for pigment formation. Since most of the steps leading to this enzyme activation are unknown, we have investigated a possible role of protein phosphorylation in the action of α-MSH on melanoma cells. Incubation of Cloudman S-91 mouse melanoma cells with 32p-phosphate results in the incorporation of 32p into a large number of phosphoproteins. In the presence of α-MSH a significant increase of 32p incorporation was observed into two phosphoproteins with apparent molecular weights of 43,000 and 34,000 daltons, respectively. This increase was concentration dependent (significant at 10−8M α-MSH) and reversible. The effect could be induced by melanotropic but not by non-melanotropic peptides. [Norleucine4, D-phenylalanine7]-α-MSH which is 100 times more active than α-MSH in stimulating tyrosinase activity, was also superactive in increasing phosphorylation of the 43,000 and 34,000 dalton proteins. These results suppose that protein phosphorylation is involved in the stimulation of melanoma cells by α-MSH.

Photoaffinity labeling of MSH receptors on pigment cells

The pituitary peptide hormone α-MSH plays a role in pigmentation and fetal development and as a neuropeptide it influences behaviour and learning. In order to study α-MSH at the molecular level we have introduced a method of photoaffinity labelling of MSH receptors on intact pigment cells with which the biological response of the labelled cells could be continuously monitored. When melanophores of skin of Anolis carolinensis or Xenopus laevis where irradiated with UV-light in the presence of photoreactive α-MSH containing the photolabel in position 1, 9, or 13, a long-lasting signal was generated inducing irreversible pigment dispersion. A number of control experiments proved that the effect was specific. Calcium was shown to be indispensable for hormone-receptor binding and for the transduction of the signal from the receptor to the adenylate cyclase. Catecholamines acting through an alpha-2 receptor could reversibly inhibit the longlasting response of Anolis melanophores. The Xenopus model was also used for studying receptor turnover and degradation in the presence and absence of calcium. These and other results showed that irreversible stimulation by covalent labelling of hormone receptors represents a useful approach for studying stimulus-response coupling in intact cells, particularly when there are only few cells available.