Cyclic lactam analogues of Ac-[Nle4]alpha-MSH4-11-NH2

Two side-chain cyclic lactam analogues of the 4-11 fragment of alpha-melanocyte-stimulating hormone (alpha-MSH), Ac-[Nle4,D-Orn5,Glu8]alpha-MSH4-11-NH2 and Ac-[Nle4,D-Orn5,D-Phe7,Glu8]alpha-MSH4-11-NH2, were prepared on p-methylbenzhydrylamine resin by using a combination of N alpha-Boc and N alpha-Fmoc synthetic strategies with diphenyl phosphorazidate mediated cyclization. The melanotropin activities of these two analogues were examined and compared relative to those of alpha-MSH, Ac-[Nle4]alpha-MSH4-11-NH2, and Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the frog (Rana pipiens) skin bioassay, the L-Phe7 17-membered ring cyclic analogue was slightly more potent than the linear Ac-[Nle4]alpha-MSH4-11-NH2 and exhibited prolonged melanotropic bioactivity (greater than or equal to 4 h). In this same assay, the D-Phe7 cyclic analogue was more than 100-fold less potent than the L-Phe cyclic analogue and was 10,000 times less potent than linear Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the lizard skin (Anolis carolinensis) bioassay, the L-Phe7 cyclic analogue was 100-fold less potent than Ac-[Nle4]alpha-MSH4-11-NH2, while the D-Phe7 cyclic analogue was 10,000-fold less potent than both Ac-[Nle4]alpha-MSH4-11-NH2 and the D-Phe7 linear derivative Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. The solution conformation of these two cyclic analogues in dimethyl sulfoxide-d6 was examined by 1D and 2D 500-MHz 1H NMR spectroscopy. Our analysis suggests an H bond stabilized C10 (or C13) turn for the D-Phe7 cyclic structure while the L-Phe7 analogue is more conformationally flexible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antagonist and agonist activities of the mouse agouti protein fragment (91-131) at the melanocortin-1 receptor.

Antagonist and agonist activities of chemically synthetized mouse agouti protein fragment (91-131) (AP91-131) at the melanocortin type-1 receptor (MC1-R) were assessed using B 16-F1 mouse melanoma cells in vitro and the following assay systems: (i) receptor binding, (ii) adenylate cyclase, (iii) tyrosinase, (iv) melanin production, and (v) cell proliferation. In competition binding studies AP91-131 was about 3-fold less potent than the natural agonist alpha-melanocyte-stimulating hormone (alpha-MSH) in displacing the radioligand [125I]-[Nle4, D-Phe7]-alpha-MSH (Ki 6.5 +/- 0.8 nmol/l). Alpha-MSH-induced tyrosinase activation and melanin production were completely inhibited by a 100-fold higher concentration of AP9 l -131; the IC50 values for AP91-131 in thetwo assay systems were 91 +/- 22 nM and 95 +/- 15 nM respectively. Basal melanin production and adenylate cyclase activity in the absence of agonist were decreased by AP91-131 with IC50 values of 9.6+/-1.8 nM and 5.0+/-2.4 nM, respectively. This indicates inverse agonist activity of AP91-131 similar to that of native AP. The presence of 10 nM melanin-concentrating hormone (MCH) slightly potentiated the inhibitory activity of AP91-131 in the adenylate cyclase and melanin assays. On the other hand, AP91-131 inhibited cell growth similar to alpha-MSH (IC50 11.0 +/- 2.1 nM; maximal inhibition 1.8-fold higher than that of alpha-MSH). Furthermore, MC1-R was down-regulated by AP91-131 with about the same potency and time-course as with alpha-MSH. These results demonstrate that AP91-131 displays both agonist and antagonist activities at the MC1-R and hence that it is the cysteine-rich region of agouti protein which inhibits and mimics the different alpha-MSH functions, most likely by simultaneous modulation of different intracellular signalling pathways.     

ACTH1-17 is a more potent agonist at the human MC1 receptor than alpha-MSH.

The melanocortin receptor MC1 is expressed on melanocytes and is an important control point for melanogenesis and other responses. Alpha-MSH, which is considered to be the major ligand at the human melanocortin (MC)1 receptor (hMC1R), is produced from proopiomelanocortin (POMC) in the pituitary and in the skin by melanocytes and keratinocytes. Other POMC peptides are also produced in the skin and their concentrations exceed those of alpha-MSH by several fold. One of the most abundant is ACTH1-17. We have shown that adrenocorticotrophic hormone (ACTH)1-17 is more potent than alpha-MSH in stimulating melanogenesis in human melanocytes and unlike alpha-MSH produces a biphasic dose response curve. In this study we have examined the ability of ACTH1-17 to function as a ligand at the hMC1R. Competitive binding assays with [125I]Nle4 DPhe7 alpha-MSH as labelled ligand were carried out in HEK 293 cells transfected with the hMC1R. ACTH1-17 showed high affinity for the hMC1R with a Ki value of 0.21 +/- 0.03 nM which was slightly higher than that of 0.13 +/- 0.005 nM for alpha-MSH. ACTH1-17 was, however, more potent than alpha-MSH in increasing cAMP and IP3 production in the transfected cells. Our results demonstrate that ACTH1-17 is a potent agonist at the hMC1R. It is therefore possible that ACTH1-17, which is found in the skin in greater concentrations than alpha-MSH, has an important role in the regulation of human melanocytes and other cell types that express the hMC1R.     

Weak functional coupling of the melanocortin-1 receptor expressed in human adipocytes

The melanocortin (MC) receptor type-1 (MC1-R) is the only one of the five MC receptor subtypes expressed in human adipose tissue explants, human mesenchymal stem cells (MSCs), and MSC-derived adipocytes. Following our recent expression studies (Obesity 2007, 15, 40-49), we now investigated the functional role of MC1-R in these tissues and cells to deduce the coupling state of MC1-R to intracellular output signals in human fat cells and tissue. Expression of MC1-R by undifferentiated and differentiated MSCs was quantified by real-time TaqMan PCR. Intracellular output signals (cAMP, lipolysis, secretion of IL-6, IL-10, and TNF-alpha), as well as effects on the metabolic rate and proliferation of human MSCs were analyzed by standard assays, exposing undifferentiated and differentiated MSCs and, in part, human adipose tissue explants to the potent MC1-R agonist, [Nle(4), D-Phe(7)]-alpha-MSH (NDP-MSH). This agonist induced a weak cAMP signal in MSC-derived adipocytes. However, it did not affect lipolysis in these cells or in adipose tissue explants, nor did it modulate cytokine release and mRNA expression of IL-6, IL-8, and TNF-alpha upon LPS stimulation. In undifferentiated MSCs, NDP-MSH did not alter the metabolic rate, but it showed a significant antiproliferative effect. Therefore, it appears that MC1-R-effector coupling in (differentiated) human adipocytes is too weak to induce a regulatory effect on lipolysis or inflammation; by contrast, MC1-R stimulation in undifferentiated MSCs induces an inhibitory signal on cell proliferation.     

Contribution of melanocortin receptor exoloops to Agouti-related protein binding.

Agouti-related protein (AGRP) is an endogenous antagonist of melanocortin action that functions in the hypothalamic control of feeding behavior. Although previous studies have shown that AGRP binds three of the five known subtypes of melanocortin receptor, the receptor domains participating in binding and the molecular interactions involved are presently unknown. The present studies were designed to examine the contribution of extracytoplasmic domains of the melanocortin-4 receptor (MC4R) to AGRP binding by making chimerical receptor constructs of the human melanocortin-1 receptor (MC1R; a receptor that is not inhibited by AGRP) and the human MC4R (a receptor that is potently inhibited by AGRP). Substitutions of the extracytoplasmic NH2 terminus and the first extracytoplasmic loop (exoloop) of the MC4R with homologous domains of the MC1R had no effect on AGRP (87-132) binding affinity or inhibitory activity (the ability to inhibit melanocortin-stimulated cAMP generation). In contrast, cassette substitutions of exoloops 2 and 3 of the MC4R with the homologous exoloops of the MC1R resulted in a substantial loss of AGRP binding affinity and inhibitory activity. Conversely, the exchange of exoloops 2 and 3 of the MC1R with the homologous exoloops of the MC4R was found to confer AGRP binding and inhibitory activity to the basic structure of the MC1R. Importantly, these substitutions did not affect the ability of the alpha-melanocyte stimulating hormone analogue [Nle4,D-Phe7] melanocyte stimulating hormone to bind or activate the chimeric receptors. These data indicate that exoloops 2 and 3 of the melanocortin receptors are important for AGRP binding.     

Anxiety-like behavior induced by IL-1beta is modulated by alpha-MSH through central melanocortin-4 receptors

The proinflammatory cytokine interleukin-1beta (IL-1beta) influences neuroendocrine activity and produces other effects, including fever and behavioral changes such as anxiety. The melanocortin neuropeptides, such as alpha-melanocyte-stimulating hormone (alpha-MSH), antagonize many actions of IL-1, including fever, anorexia and hypothalamic-pituitary-adrenal (HPA) axis activation through specific melanocortin receptors (MC-R) in the central nervous system. The objective of the present study was to establish the effect of MSH peptides on IL-1beta-induced anxiety-like behavior and the melanocortin receptors involved. We evaluated the effects of intracerebroventricular (i.c.v.) administration of IL-1beta (30 ng) and melanocortin receptor agonists: alpha-MSH, an MC3/MC4-R agonist (0.2 microg) or gamma-MSH, an MC3-R agonist (2 microg) or HS014, an MC4-R antagonist (2 microg), on an elevated plus-maze (EPM) test. Injection of IL-1beta induced an anxiogenic-like response, as indicated by reduced open arms entries and time spent on open arms. The administration of alpha-MSH reversed IL-1beta-induced anxiety with co-administration of HS014 inhibiting the effect of alpha-MSH. However, the associated treatment with gamma-MSH did not affect the anxiety response to IL-1beta. These data suggest that alpha-MSH, through central MC4-R can modulate the anxiety-like behavior induced by IL-1beta.     

Use of an alpha-melanocyte-stimulating hormone analogue to improve alpha-melanocyte-stimulating hormone receptor binding assay in human melanoma

In order to optimize the detection and measurement of alpha-melanocyte-stimulating hormone (alpha-MSH) receptivity in human melanoma cells, and the authors replaced the natural hormone by [Nle4,D-Phe7]-alpha-MSH, a more stable and potent analogue in the receptor binding assay commonly performed with alpha-MSH. The following parameters were investigated: temperature, incubation time, number of cells, and ratio of labelled to unlabelled hormone. Optimal conditions for each assay were determined. The results demonstrate that the analogue has identical binding sites to alpha-MSH, as similar reciprocal displacements of each labelled (125I) hormone by serial dilutions of unlabelled alpha-MSH or [Nle4,D-Phe7]-alpha-MSH (10(-12) M to 10(-6) M) were obtained. To further compare the two hormones, we performed a screening of various human cell lines: ten melanomas and five nonmelanomas. The assay with [Nle4,D-Phe7]-alpha-MSH yielded more receptor expression on six of ten melanoma lines against only four of ten with the natural hormone. In conclusion, the use of radiolabelled [Nle4,D-Phe7]-alpha-MSH analogue instead of labelled alpha-MSH improved both sensitivity and reproducibility in this receptor binding assay on human melanoma lines.     

Behavioral effects of [4-norleucine, 7-D-phenylalanine]-alpha-melanocyte-stimulating hormone

The behavioral effects of alpha-melanocyte stimulating hormone (alpha-MSH) were compared to an alpha-MSH analogue that had a norleucine substituted for methionine in the four position and a D-phenylalanine substituted for L-phenylalanine in the seven position. [Nle4, D-Phe7]-alpha-MSH has previously been shown to be a superpotent agonist on melanocytes [17]. The present experiments indicate that [Nle4, D-Phe7]-alpha-MSH is equipotent to alpha-MSH in inducing grooming when administered intraventricularly. In contrast, the analogue has the opposite effect of alpha-MSH on performance of a visual discrimination task. alpha-MSH improves visual performance whereas [Nle4, D-Phe7]-alpha-MSH attenuates such performance. The contrasting activities of [Nle4, D-Phe7]-alpha-MSH on the physiological processes described suggest that this analogue may interact with three distinct melanotropin receptors in different ways. On melanocyte receptors the melanotropin analogue is a superagonist, on CNS melanotropin receptors involved in grooming it is equipotent to alpha-MSH, and on CNS receptors involved in attention, learning and memory [Nle4, D-Phe7]-alpha-MSH may be an antagonist of endogenous melanotropin.     

Conformation of the core sequence in melanocortin peptides directs selectivity for the melanocortin MC3 and MC4 receptors.

Melanocortin peptides regulate a variety of physiological processes. Five melanocortin receptors (MC-R) have been cloned and the MC3R and MC4R are the main brain MC receptors. The aim of this study was to identify structural requirements in both ligand and receptor that determine gamma-melanocyte-stimulating hormone (MSH) selectivity for the MC3R versus the MC4R. Substitution of Asp10 in [Nle4]Lys-gamma2-MSH for Gly10 from [Nle4]alpha-MSH, increased both activity and affinity for the MC4R while the MC3R remained unaffected. Analysis of chimeric MC3R/MC4Rs and mutant MC4Rs showed that Tyr268 of the MC4R mainly determined the low affinity for [Nle4]Lys-gamma2-MSH. The data demonstrate that Asp10 determines selectivity for the MC3R, however, not through direct side chain interactions, but probably by influencing how the melanocortin core sequence is presented to the receptor-binding pocket. This is supported by mutagenesis of Tyr268 to Ile in the MC4R which increased affinity and activity for [Nle4]Lys-gamma2-MSH, but decreased affinity for two peptides with constrained cyclic structure of the melanocortin core sequence, MT-II and [D-Tyr4]MT-II, that also displayed lower affinity for the MC3R. This study provides a general concept for peptide receptor selectivity, in which the major determinant for a selective receptor interaction is the conformational presentation of the core sequence in related peptides to the receptor-binding pocket.     

beta-MSH: a functional ligand that regulated energy homeostasis via hypothalamic MC4-R?

alpha-Melanocyte stimulating hormone (MSH) has generally been assumed to be the endogenous ligand acting at the melanocortin-4 receptor (MC4-R), activation of which in the hypothalamus leads to reduced feeding. However, beta-MSH is also capable of activating MC4-R and inhibiting feeding. Here, we investigated the possibility that beta-MSH acts as an endogenous MC4-R agonist and that this melanocortin peptide plays a role in the regulation of feeding and energy balance. We found that beta-MSH had significantly higher affinities than alpha-MSH at both human MC4-R transfected into CHO cells (K(i): beta-MSH, 11.4+/-0.4 nmol/l versus alpha-MSH, 324+/-16 nmol/l, P<0.001) and MC4-R in rat hypothalamic homogenates (K(i): beta-MSH, 5.0+/-0.4 nmol/l versus alpha-MSH, 22.5+/-2.3 nmol/l, P<0.001). Incubation of brain slices with 5 microM beta-MSH significantly increased [35S]GTPgammaS binding by 140-160% (P<0.001), indicating activation of G-protein-coupled receptors (GPCRs), in the hypothalamic ventromedial (VMH), dorsomedial (DMH), arcuate (ARC) and paraventricular (PVN) nuclei. These sites match the distribution of beta-MSH immunoreactive fibres and also the distribution of MC4-R binding sites which we and others previously reported. Food-restriction significantly increased beta-MSH levels in the VMH, DMH and ARC (all P<0.05) above freely-fed controls, whilst alpha-MSH concentrations were unchanged. We propose that increased beta-MSH concentrations reflect blockade of the peptide's release in these sites, consistent with the increased hunger and the known up-regulation of MC4-R in the same nuclei. Thus, we conclude that (1). beta-MSH has higher affinity at MC4-R than alpha-MSH; (2). beta-MSH activates GPCR in these sites, which are rich in MC4-R; and (3). beta-MSH is present in hypothalamic nuclei that regulate feeding and its concentrations alter with nutritional state. We suggest that beta-MSH rather than alpha-MSH is the key ligand at the MC4-R populations that regulate feeding, and that inhibition of tonic release of beta-MSH is one mechanism contributing to hunger in under-feeding.     

Peripheral effect of alpha-melanocyte-stimulating hormone on fatty acid oxidation in skeletal muscle

To study the peripheral effects of melanocortin on fuel homeostasis in skeletal muscle, we assessed palmitate oxidation and AMP kinase activity in alpha-melanocyte-stimulating hormone (alpha-MSH)-treated muscle cells. After alpha-MSH treatment, carnitine palmitoyltransferase-1 and fatty acid oxidation (FAO) increased in a dose-dependent manner. A strong melanocortin agonist, NDP-MSH, also stimulated FAO in primary culture muscle cells and C2C12 cells. However, [Glu6]alpha-MSH-ND, which has ample MC4R and MC3R agonistic activity, stimulated FAO only at high concentrations (10(-5) M). JKC-363, a selective MC4R antagonist, did not suppress alpha-MSH-induced FAO. Meanwhile, SHU9119, which has both antagonistic activity on MC3R and MC4R and agonistic activity on both MC1R and MC5R, increased the effect of alpha-MSH on FAO in both C2C12 and primary muscle cells. Small interference RNA against MC5R suppressed the alpha-MSH-induced FAO effectively. cAMP analogues mimicked the effect of alpha-MSH on FAO, and the effects of both alpha-MSH and cAMP analogue-mediated FAO were antagonized by a protein kinase A inhibitor (H89) and a cAMP antagonist ((Rp)-cAMP). Acetyl-CoA carboxylase activity was suppressed by alpha-MSH and cAMP analogues by phosphorylation through AMP-activated protein kinase activation in C2C12 cells. Taken together, these results suggest that alpha-MSH increases FAO in skeletal muscle, in which MC5R may play a major role. Furthermore, these results suggest that alpha-MSH-induced FAO involves cAMP-protein kinase A-mediated AMP-activated protein kinase activation.     

Type I beta-turn conformation is important for biological activity of the melanocyte-stimulating hormone analogues.

In order to define which structure of alpha-melanocyte-stimulating hormone (MSH) analogues plays a critical role for ligand-receptor interaction and selectivity, we analysed receptor-binding and cAMP-generating activity in Chinese hamster ovary cell lines stably transfected with rMC3R and hMC4R, as well as the NMR structures of chemically synthesized alpha-MSH analogues. Compared with [Ahx4]alpha-MSH, the linear MTII designated as alpha-MSH-ND revealed a preference for the MC4R, whereas its IC50 and EC50 values were comparable to those of MTII reported previously. Truncation of Ahx4 and Asp5 of alpha-MSH-ND remarkably decreased the receptor-binding and cAMP-generating activity. Meanwhile, maximum cAMP-generating activity was observed at a higher concentration (10(-5) M) of alpha-MSH-ND(6-10), and MC4R preference was changed into MC3R preference. In contrast, [Gln6]alpha-MSH-ND(6-10) lost its cAMP-generating activity almost completely, even though it bound to both receptors. Whereas the solution conformation of alpha-MSH-ND revealed a stable type I beta-turn structure, [Gln6]alpha-MSH-ND(6-10) revealed a tight gamma-turn composed of Gln6-D-Phe7-Arg8. Replacement of the His6 residue of alpha-MSH-ND by Gln, Asn, Arg or Lys decreased not only the receptor binding, but also the cAMP-generating activity in both the MC3R and the MC4R. The structure of [Gln6]alpha-MSH-ND exhibited a stable type I' beta-turn comprising Asp5, Gln6, D-Phe7 and Arg8. [Lys6]alpha-MSH-ND showed a greatly reduced binding affinity and cAMP-generating activity with the loss of MC4R selectivity. In NMR studies, [Lys6]alpha-MSH-ND also demonstrated a gamma-turn conformation around Lys6-DPhe7-Arg8. From the above results, we conclude that a type I beta-turn conformation comprising the residues Asp5-His6-(D-Phe7)-Arg8 was important for receptor binding and activation, as well as the selectivity of MSH analogues.     

Agouti-related protein has an inhibitory paracrine role in the rat adrenal gland

alpha-Melanocyte-stimulating-hormone (alpha-MSH) is an agonist at the melanocortin 3 receptor (MC3-R) and melanocortin 4 receptor (MC4-R). alpha-MSH stimulates corticosterone release from rat adrenal glomerulosa cells in vitro. Agouti-related protein (AgRP) an endogenous antagonist at the MC3-R and MC4-R, is expressed in the adrenal gland. We investigated the expression of the MC3-R and MC4-R and the role of AgRP in the adrenal gland. MC3-R and MC4-R expression was detected in rat adrenal gland using RT-PCR. The effect of AgRP on alpha-MSH-induced corticosterone release was investigated using dispersed rat adrenal glomerulosa cells. AgRP administered alone did not affect corticosterone release, but co-administration of AgRP and alpha-MSH attenuated alpha-MSH-induced corticosterone release. To investigate glucocorticoid feedback, adrenal AgRP expression was compared in rats treated with dexamethasone to controls. AgRP mRNA was increased in rats treated with dexamethasone treatment compared to controls. Our findings demonstrate that adrenal AgRP mRNA is regulated by glucocorticoids. AgRP acting via the MC3-R or MC4-R may have an inhibitory paracrine role, blocking alpha-MSH-induced corticosterone secretion.     

Potent and prolonged melanotropic activities of the alpha-MSH fragment analog, Ac-[Nle4,D-Phe7]-alpha-MSH4-9-NH2

Ac-[Nle4, D-Phe7]-alpha-MSH4-9-NH2 and Ac-[Nle4]-alpha-MSH4-9-NH2, fragment analogs of the tridecapeptide, alpha-melanocyte stimulating hormone (alpha-MSH, alpha-melanotropin), were synthesized. The potency and prolonged activity of the analogs were compared to alpha-MSH in several melanotropin bioassays. The D-Phe-containing hexapeptide was 10 times more active than alpha-MSH in stimulating melanoma tyrosinase activity. This analog was also 10-fold more potent than alpha-MSH in the lizard skin bioassay and about 10-fold less active in the frog skin bioassay. The melanotropic activity of Ac-[Nle4, D-Phe7]-alpha-MSH4-9-NH2 was considerably prolonged compared to alpha-MSH in each of the bioassays. These results demonstrate that the structural requirements for superpotency and prolonged activity of [Nle4, D-Phe7]-substituted analogs reside within this hexapeptide sequence.     

Synthesis and receptor binding analysis of thirteen oligomeric alpha-MSH analogs

Thirteen oligomeric analogs from dimers up to a hexamer of alpha-melanocyte-stimulating hormone (alpha-MSH) were synthesized and tested on melanoma cells for their ability to bind to melanocortin type 1 (MC1) receptors and to stimulate melanin production in the cells. The peptidic oligomers were made by linking several copies of the alpha-MSH fragment analog Nle-Asp-His-[D-Phe]-Arg-Trp-Lys-NH2 to different templates through formation of oxime bonds. They were found to have binding affinities at 37 degrees C up to 8 times higher and melanogenesis-inducing activities up to 4 times higher than those of the native hormone. At 15 degrees C, one dimer showed a binding affinity 20 times higher than that of alpha-MSH. These results are discussed in terms of possible bridging of neighboring receptors which has been suggested to occur in some other systems.     

Cysteine residues are involved in structure and function of melanocortin 1 receptor: Substitution of a cysteine residue in transmembrane segment two converts an agonist to antagonist

Reduction of disulfide bonds in human melanocortin 1 receptor (hMC1R) with increasing concentrations of DTT (dithiothreitol) resulted in a decrease in the binding of [125I]-ACTH (adrenocorticotropic hormone, L-isomer) in an uniphasic manner and a decrease in [125I]-NDP-MSH ([Nle(4),D-Phe(7)]-alpha-melanocyte stimulating hormone; D-isomer) binding in a biphasic manner. Pretreatment of hMC1R with 10 mM DTT resulted in a 36-fold loss of affinity for alpha-MSH (L-isomer) without affecting the affinity of NDP-MSH (D-isomer). To characterize the role of individual cysteine residues, we employed site-directed mutagenesis to substitute cysteine by glycine at all fourteen positions in hMC1R and analysed wild-type and mutant receptors for ligand binding and cAMP signalling. Single point mutation of four cysteine residues in extracellular loops to glycine (C35G, C267G, C273G, and C275G) resulted in a complete loss of binding for [125I]-NDP-MSH. Moreover, mutants with normal ligand binding, at positions C191G (transmembrane segment 5), C215G (third intracellular loop), and C315G (C-terminal loop) failed to generate cAMP signal in response to both agonists alpha-MSH and NDP-MSH. Mutant at position C78G (with wild-type binding to alpha-MSH as well as NDP-MSH) generated a cAMP signal in response to alpha-MSH (identical to wild-type hMC1R) but interestingly could not be stimulated by NDP-MSH. Moreover, this single amino acid substitution converted NDP-MSH from being an agonist to antagonist at the C78G mutant receptor. These findings demonstrate that (i) alpha-MSH and ACTH (L-isomers) are different from D-isomer NDP-MSH in their sensitivity to DTT for receptor binding, (ii) cysteine residues in N-terminus and extracellular loop three make disulfide bridges and are needed for structural integrity of hMC1R, (iii) cysteine residues in transmembrane segments and intracellular loops are required for receptor-G-protein coupling, (iv) C78 in transmembrane segment two is required for generating a functional response by D-isomer agonist (NDP-MSH) but not by L-isomer agonist (alpha-MSH), and (v) wild-type receptor agonist NDP-MSH is an antagonist at the mutant C78G receptor.     

alpha-MSH analogues and adrenal zona glomerulosa function

Recent studies on the control of adrenal zona glomerulosa function and aldosterone secretion have focussed attention on the role of MSH-like peptides. In particular, at low concentrations, alpha-MSH has a specific stimulatory effect on rat adrenal glomerulosa cells. The synthesis of alpha-MSH analogues which have potent and prolonged effects on melanocyte systems offers new methods of examining the specificity of this response. Two peptides were tested in which potential for a beta-turn configuration was stabilised. These were: [Nle4, D-Phe7]-alpha-MSH and the cyclic [Cys4, Cys10]-alpha-MSH. In contrast to their effects on melanocyte systems, only [Cys4, Cys10]-alpha-MSH stimulated glomerulosa cells, and it was equipotent with alpha-MSH, while [Nle4, D-Phe7]-alpha-MSH and shorter fragments had no effect when added alone. [Nle4, D-Phe7]-alpha-MSH, however, augmented the response of cells already maximally stimulated with alpha-MSH and in this respect its actions resembled those of gamma-MSH and related peptides. The augmentation produced by [Nle4, D-Phe7]-alpha-MSH and gamma 3-MSH was not additive when the two peptides were added together with alpha-MSH. The results suggest that the specificity of the alpha-MSH receptors in rat adrenal glomerulosa cells and the peptide structure-function relationships in this system are different from those described for melanocytes.     

Relative stability of alpha-melanotropin and related analogues to rat brain homogenates

alpha-Melanotropin (alpha-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle4,D-Phe7]-alpha-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle4,D-Phe7]-alpha-MSH4-10-NH2, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle4,D-Phe7]-alpha-MSH4-11-NH2 is totally resistant to inactivation by rat brain homogenate. Both [Nle4,D-Phe7]-fragments are resistant to degradation by rat serum, but [Nle4]-alpha-MSH, Ac-[Nle4]-alpha-MSH4-10-NH2 and Ac-[Nle4]-alpha-MSH4-11-NH2 are rapidly inactivated under both conditions. The cyclic melanotropin, [Cys4,Cys10]-alpha-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[Cys4,Cys10]-alpha-MSH4-10-NH2 analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-10-NH2 is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues.     

Molecular determinants of ligand binding to the human melanocortin-4 receptor

To elucidate the molecular basis for the interaction of ligands with the human melanocortin-4 receptor (hMC4R), agonist structure-activity studies and receptor point mutagenesis were performed. Structure-activity studies of [Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) identified D-Phe7-Arg8-Trp9 as the minimal NDP-MSH fragment that possesses full agonist efficacy at the hMC4R. In an effort to identify receptor residues that might interact with amino acids in this tripeptide sequence 24 hMC4R transmembrane (TM) residues were mutated (the rationale for choosing specific receptor residues for mutation is outlined in the Results section). Mutation of TM3 residues D122 and D126 and TM6 residues F261 and H264 decreased the binding affinity of NDP-MSH 5-fold or greater, thereby identifying these receptor residues as sites potentially involved in the sought after ligand-receptor interactions. By examination of the binding affinities and potencies of substituted NDP-MSH peptides at receptor mutants, evidence was found that core melanocortin peptide residue Arg8 interacts at a molecular level with hMC4R TM3 residue D122. TM3 mutations were also observed to decrease the binding of hMC4R antagonists. Notably, mutation of TM3 residue D126 to alanine decreased the binding affinity of AGRP (87-132), a C-terminal derivative of the endogenous melanocortin antagonist, 8-fold, and simultaneous mutations D122A/D126A completely abolished AGRP (87-132) binding. In addition, mutation of TM3 residue D122 or D126 decreased the binding affinity of hMC4R antagonist SHU 9119. These results provide further insight into the molecular determinants of hMC4R ligand binding.     

Biological activity, binding, and metabolic fate of Ac-[Nle4, D-Phe7]alpha-MSH4-11NH2 with the F1 variant of B16 melanoma cells

The alpha-MSH (alpha-melanocyte-stimulating hormone) agonist, Ac-[Nle4, D-Phe7]alpha-MSH4-11NH2 (hereafter called ND4-11 alpha-MSH), is at least 10-fold more potent than alpha-MSH as a stimulus of tyrosinase activity in F1 variant cells of B16 melanoma. The binding to these cells during an incubation with 5 nM (3H)ND4-11 alpha-MSH at 37 degrees C is maximal at 0-30 min, 22 fmol/10(6) cells, but declines to 40% of this value at 4 hr. in the presence of 5 nM (3H)ND4-11 alpha-MSH at 37 degrees C, the acid soluble (cell surface) radioactivity decreased rapidly from 11.4 fmol/10(6) cells at 5 min to 4.6 fmol/10(6) cells at 4 hr. Chromatographic analysis of media and cellular samples revealed that there was no evidence of degradation of (3H)ND4-11 alpha-MSH in the medium but there was evidence of intracellular degradation of (3H)ND4-11 alpha-MSH. Ammonium chloride (10mM) resulted in an increase in acid resistant radioactivity (internalized hormone) at 4 hr. The binding to F1 variant cells during an incubation with 0.155 nM or 5 nM (3H)ND4-11 alpha-MSH at 4 degrees C was constant from 4 hr to 24 hr. Under these conditions, there was no time-dependent change in the acid soluble radioactivity from 4 to 24 hr. Scatchard analysis of (3H)ND4-11 alpha-MSH binding to F1 variant cells at 4 degrees C demonstrated that there were approximately 4500 receptors per cell and an association constant of 17.1 nM-1. These results are consistent with a process of (3H)ND4-11 alpha-MSH binding to its receptor followed by internalization of the receptor-hormone complex and then intracellular degradation of the hormone.