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.     

In situ melanin assay for MSH using mouse B16 melanoma cells in culture

A sensitive in situ melanin assay using cultured mouse B16 melanoma cells is described for structure-activity studies with melanocyte-stimulating hormone (MSH) peptides. B16 Cells were seeded at a density of 2500 cells per well in 96-well microtest tissue culture plates; after 24 h the cells were incubated in the presence of serial dilutions of MSH peptides for 3 to 5 days. The melanin released into the medium of each well was then determined spectrophotometrically at a wavelength of 405 nm using an automatic microplate reader calibrated against synthetic melanin. Studies with alpha-MSH, [Nle4, D-Phe7]-alpha-MSH, [3'-iodo-Tyr2]-alpha-MSH, adrenocorticotropin (ACTH)(1-24), and ACTH(1-39) showed that the peptides had identical intrinsic activities and that the relative potencies were similar to those obtained with a tyrosinase assay. The EC50 of alpha-MSH was 27 pM, i.e., about five- to sevenfold lower than that in the assays for tyrosinase or intracellular melanin. Thus, the new assay represents the most sensitive melanoma cell assay for MSH available to date.     

POMC-derived peptides in the neuromuscular system of streptozotocin-diabetic mice.

Immunoreactivity for beta-endorphin and alpha-MSH/ACTH was demonstrated in intramuscular nerves in soleus, extensor digitorum longus, and diaphragm muscles of normal and streptozotocin-diabetic mice. There was a higher incidence of immunoreactive nerves in the muscles of the diabetic mice. Specific binding for [125I]ACTH was detected in a proportion of the muscle fibers, using autoradiography. There were significantly more fibers with specific [125I]beta-endorphin sites and specific [125]ACTH sites in some muscles in the diabetic mice. The increased expression of POMC-derived peptides and their receptors in the neuromuscular system of streptozotocin-diabetic mice may indicate early neuropathic change.     

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.     

Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA.

Melanocytes and melanoma cells are known to possess receptors for melanocyte stimulating hormone (MSH). A cDNA clone, designated 11D, has been isolated from human melanoma cells and encodes a MSH receptor. The cloned cDNA encodes a 317 amino acid protein with transmembrane topography characteristics of a G-protein-coupled receptor, but it does not show striking similarity to already published sequences of other G-protein-coupled receptors. When 11D cDNA is expressed in COS-7 cells, it binds an 125I-labelled MSH analogue (NDP-MSH) in a specific manner. The bound ligand could be displaced by melanotropic peptides, alpha-MSH, beta-MSH, gamma-MSH and ACTH (adrenocorticotropic hormone), but not by the non-melanotropic peptide, beta-endorphin. This is the first report of the cloning of the receptor gene of the melanotropin receptor family.     

Interaction of melanin-concentrating hormone (MCH), neuropeptide E-I (NEI), neuropeptide G-E (NGE), and alpha-MSH with melanocortin and MCH receptors on mouse B16 melanoma cells.

Melanin-concentrating hormone (MCH) and alpha-melanocyte-stimulating hormone (alpha-MSH) are known to exhibit mostly functionally antagonistic, but in some cases agonistic activities, e.g., in pigment cells and in the brain. Neuropeptide E-I (NEI) displays functional MCH-antagonist and MSH-agonist activity in different behavioral paradigms; the role of neuropeptide G-E (NGE) is not known. This study addressed the question of possible molecular interactions between alpha-MSH, MCH and the MCH-precursor-derived peptides NEI and NGE at the level of the pigment cell MCH receptor subtype (MCH-Rpc) and the different melanocortin (MC) receptors. Radioreceptor assays using [125I]MCH, [125l]alpha-MSH and [125I]NEI as radioligands and bioassays were performed with MCI-R-positive and MC1-R-negative mouse B16 melanoma cells and with COS cells expressing the different MC receptors. The IC50s of alpha-MSH and NEI or NGE for [125I]MCH displacement from mouse MCH-Rpc were 80-fold and, respectively, >300-fold higher than that of MCH, and the IC50s for MCH and NEI or NGE for [125I]alpha-MSH displacement from mouse MC1-R were 50,000-fold and >200,000-fold higher than that of alpha-MSH. No high-affinity binding sites for NEI were detected on B16 melanoma cells and there was no significant displacement of [1251]alpha-MSH by MCH, NEI or NGE with MC3-R, MC4-R and MC5-R expressed in COS cells. At concentrations of 100 nM to 10 microM, however, MCH, NEI and NGE induced cAMP formation and melanin synthesis which could be blocked by agouti protein or inhibitors of adenylate cyclase or protein kinase A. This shows that mammalian MCH-precursor-derived peptides may mimic MSH signalling via MC1-R activation at relatively high, but physiologically still relevant concentrations, as e.g. found in autocrine/paracrine signalling mechanisms.     

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.     

Kinetic evidence for tandemly arranged ligand binding sites in melanocortin 4 receptor complexes

The melanocortin 4 receptor (MC(4)R) binding of the peptide analogue of melanocyte stimulating hormone, [(125)I]NDP-MSH, and the low molecular weight radionucleid 1-(D-1,2,3,4-tetrahydroisoquinoline-3-carboxy-D-4-(125)iodophenylalanyl)-4-cyclohexyl-4-[(1,2,4-triazol-1-yl)methyl]piperidine trifluoroacetate ([(125)I]THIQ) were compared. Kinetic analysis indicated heterogeneity in the binding of both radioligands, the binding apparently proceeding to two tandemly arranged interconnected mutually dependent binding sites. Steric considerations and BRET analysis of Rluc and GFP tagged receptors proposed that these sites are located on different subunits of receptor dimers, which form receptor complexes. According to the minimal model proposed, ligand binding proceeds consecutively to the two binding sites of the dimer. After binding of the first ligand conformational transformations of the complex occur, which is followed by binding of the second ligand. When both receptor units have bound [(125)I]NDP-MSH, the radioligand can be released only from one unit. The [(125)I]NDP-MSH bound to the remaining unit stays practically irreversibly bound due to a very slow retransformation rate of the transformed complex. The considerably faster binding of [(125)I]THIQ did not allow accurate kinetic differentiation of the two binding sites. However, addition of NDP-MSH as well as a fragment of the human agouti protein, hAGRP(83-132) to the preformed [(125)I]THIQ-MC(4)R complex drastically retarded the release of [(125)I]THIQ from the complex, blocking conformational transformations in the complex by binding into the second binding site. The consecutive binding of ligands to the MC(4)R dimers has substantial impact on the apparent ligand potencies, when determined in competition with the two different radioligands applied herein; the apparent potencies of the same ligand differing up to three orders of magnitude when assayed in competition with [(125)I]NDP-MSH or [(125)I]THIQ.     

Comparison of structural requirements of alpha-MSH and ACTH for inducing excessive grooming and pigment dispersion

alpha-MSH and ACTH-like peptides are known to play an important role in the adaptation of many vertebrates to a new environment. These peptides induce pigment dispersion in amphibian melanophores through a receptor-mediated mechanism. In this study we compared the structural requirements of these peptides for melanotropic activity on Xenopus laevis melanophores with those for inducing excessive grooming in the rat. With the exception of ACTH1-24 there is a close resemblance in structure-activity relationships of the fragments and analogs tested in the two bioassays. [Nle4,-D-Phe7]-alpha-MSH is extremely active in both assays. Weak agonists such as [Leu9]-alpha-MSH did not possess antagonistic properties either in the melanophore assay or in the excessive grooming test. The data suggest that the mechanism of action of alpha-MSH-like peptides in rat brain is receptor-mediated like their action on melanophores.     

Neuropeptide Y receptor in cultured vascular smooth muscle cells: ligand binding and increase in cytosolic free Ca2+

We have previously shown that neuropeptide Y (NPY) increases cytosolic free Ca2+ concentration [( Ca2+]i) in porcine aortic smooth muscle cells. In this study, specific NPY receptor binding sites were identified in the cells by use of [125I]Bolton-Hunter NPY [( 125I]BH-NPY). Binding was to a single population of the sites with a Kd of 1.1 +/- 0.2 nM and a Bmax of 0.68 +/- 0.10 pmol/mg protein. [125I]BH-NPY binding was displaced by NPY-related peptides including members of the pancreatic polypeptide (PP) family. The potency of these peptides other than human PP for displacing [125I]BH-NPY binding was substantially consistent with their potency for increasing [Ca2+]i. Human PP had no effect on [Ca2+]i even at 10(-5) M, but it inhibited the NPY-induced increase in [Ca2+]i with a potency comparable to that for displacing [125I]BH-NPY binding. NPY(13-36) was about 500 and 300 times less effective than porcine NPY in increasing [Ca2+]i and in displacing [125I]BH-NPY binding, respectively, showing that the NPY receptor in cultured vascular smooth muscle cells is of the Y1-type.     

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.     

Comparative biological activities of potent active-site analogues of alpha-melanotropin. Effect of tyrosine substitution at position-4

We have prepared several alpha-melanotropin (alpha-MSH) analogues with tyrosine substituted for methionine at the 4-position and determined their melanotropic activities on the frog (Rana pipiens), lizard (Anolis carolinensis) and S-91 (Cloudman) mouse melanoma adenylate cyclase bioassays. The potencies of Ac-[Tyr4]-alpha-MSH4-10-NH2 and Ac-[Tyr4]-alpha-MSH4-11-NH2 were compared with alpha-MSH and with their corresponding methionine and norleucine substituted analogues. The Tyr-4 analogues were found to be less active than the Nle-4 analogues on both the frog and lizard assays. Ac-[Tyr4]-alpha-MSH4-10-NH2 was found to be less active than Ac-[Tyr4]-alpha-MSH4-11-NH2 on the lizard bioassay, but more active than the longer fragment on the frog skin assay. Ac-[Tyr4]-alpha-MSH4-10-NH2 exhibited extremely prolonged biological activity on frog skin, but not on lizard skin, while the melanotropic activity of Ac-[Tyr4]-alpha-MSH4-11-NH2 was rapidly reversed on both assay systems. The increased potency of Ac-[Tyr4]-alpha-MSH4-10-NH2 over Ac-[Tyr4]-alpha-MSH4-11-NH2 on frog melanocytes may be related to the fact that the shorter 4-10 analogue exhibits prolonged biological activity. Interestingly, it was found that both Tyr-4 analogues were partial agonists on the mouse melanoma adenylate cyclase bioassay, and stimulated the enzyme to only about 50% of the maximal activity of alpha-MSH. We reported previously that replacement of L-Phe-7 by its D-enantiomer in [Nle4]-alpha-MSH and its Nle-4 containing analogues resulted in peptides with increased potency and in some instances prolonged activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of gene delivery by an analogue of alpha-MSH in a receptor-independent fashion

In order to transfect melanoma specifically by receptor-mediated endocytosis we prepared dioctadecyl aminoglycylspermine (lipospermine)--DNA complexes with [Nle(4),D-Phe(7)]-alpha-MSH(4--10), a pseudo-peptide analogue of alpha-melanocyte stimulating hormone (alpha-MSH) linked to a thiol-reactive phospholipid. With these complexes we obtained an up to 70-fold increase of transfection with B16-F1 melanoma cells. However when B16-G4F, an alpha-MSH receptor negative melanoma cell line was transfected, an up to 700-fold increased transfection efficiency was observed. The peptide hormone analogue was equally efficient when it was only mixed with lipospermine--DNA complexes without covalent coupling. In addition to melanoma cells we also obtained up to 30-fold increased transfection with BN cells (embryonic liver cells). Our data show that an alpha-MSH analogue increased transfection independently of the MSH receptor expression but reaches efficiencies approaching those obtained with peptides derived from viral fusion proteins. The absence of targeting of constructs containing [Nle(4),D-Phe(7)]-alpha-MSH(4-10) can probably be attributed due to the relatively modest number of MSH receptors at the surface of melanoma. We suggest, however, that the peptide hormone analogue used in this study has membrane-active properties and could be of interest as helper agent to enhance non-viral gene delivery presumably by endosomal-destabilizing properties.     

Characterization of specific receptors for atrial natriuretic factor in bovine adrenal zona glomerulosa

We have recently shown that synthetic rat atrial natriuretic factor (ANF) directly inhibits mineralocorticoid and glucocorticoid secretion in cultured bovine adrenal cells with a potency of 100 pM. [125I]iodo-ANF was used in the present study to characterize potential receptor sites in bovine zona glomerulosa membranes. ANF binds to a class of high affinity binding sites with a pK of 10.2 and a density of 1.3 pmol/mg protein. Detailed competition curves with ANF document a class of high affinity sites with a pK of 10.2 and also a second class of lower affinity sites with a pK of 8.5. Nonspecific binding amounts to less than 10% of [125I]iodo-ANF binding at concentrations less than 100 pM. High affinity binding of [125I]iodo-ANF is reversible with a half-time of association of 15 minutes at 25 pM and a half-time of dissociation of 140 minutes. Monovalent cations Na, Li and K equipotently enhance [125I]iodo-ANF specific binding. Divalent cations Mg, Ca and Mn also increase [125I]iodo-ANF specific binding, with Mn being the most active cation. No effect of guanine nucleotide could be detected on ANF binding. The binding of [125I]iodo-ANF is very specific and is not inhibited by 1 microM angiotensin II, ACTH, VIP, somatostatin, Leu-enkephalin, dynorphin or by the N-terminal of POMC. The N-terminal fragment ANF-(1-16) is also completely inactive. Reduction of the disulfide bridge of ANF inactivates the peptide. This enabled the development of a highly specific radio-receptor assay for ANF with a minimum detectable dose of 2 femtomoles. The results document the specific receptor involved in the potent inhibitory effect of ANF on adrenal steroidogenesis and indicate that bovine adrenal zonal glomerulosa provide a highly sensitive system for studying the recently discovered atrial natriuretic factor.     

Evidence for increased alpha MSH receptor binding in the F1 variant of B16 melanoma cells grown in dialyzed fetal calf serum

The specific binding of an alpha MSH analogue (Ac-[Nle4, D-Phe7] alpha MSH4-11 NH2) was enhanced in the presence of 10% dialyzed fetal calf serum (FCS) as compared with 10% FCS (nondialyzed) in the F1 variant of B16 melanoma cells. The replenishment of dialyzed serum with adrenocorticotrophic hormone (ACTH) or insulin had no effect on the increased level of alpha MSH receptor binding in these cells. However, 10 nM alpha MSH or 1 microM ACTH under identical conditions significantly decreased the level of alpha MSH binding. Competitive binding studies involving the alpha MSH analogue revealed that the specificity of the receptor was restricted to the complete molecule of alpha MSH, our analogue, beta MSH and ACTH1-24, ACTH4-10, which contains the amino acid sequence responsible for biological activity, showed a very low affinity for the receptor. Furthermore, we observed an interesting phenomenon unique to dialyzed FCS in that once the cells were grown to confluence and melanin was produced, the cells were no longer viable. However, in McCoy's medium, which is deficient in tyrosine, the cells did not produce melanin and remained viable.     

Comparative affinities of adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) for [125I] AM and [125I] CGRP specific binding sites in porcine tissues.

We have investigated the binding characteristics of rat [125I] adrenomedullin (AM) and human [125I] calcitonin gene-related peptide (CGRP) to membranes prepared from a number of porcine tissues including atrium, ventricle, lung, spleen, liver, renal cortex and medulla. These membranes displayed specific, high affinity binding for [125I] rat AM and [125I] human CGRP. Porcine lung displayed the highest density of binding sites for radiolabeled AM and CGRP followed by porcine renal cortex. Competition experiments performed with [125I] rat AM indicated that the rank order of potencies of various peptides for inhibiting [125I] rat AM binding to various tissues were rat AM > or = human AM > or = human AM(22-52) > h alpha CGRP > or = h alpha CGRP(8-37) > sCT except spleen, atrium, renal cortex and renal medulla where rAM and hAM were 20-300 fold more potent than hAM (22-52). When the same experiments were performed using [125I] h alpha CGRP as the radioligand, the rank order potencies for various peptides were rAM = hAM > h alpha CGRP > h alpha CGRP(8-37) in most of the tissues except in spleen and liver where h alpha CGRP was the most potent ligand. In lung, h alpha CGRP was almost as potent as rAM and hAM in displacing [125I] h alpha CGRP binding. These data suggest the existence of distinct CGRP and AM specific binding sites in contrast to previous reports that showed that both peptides interact differently in rat tissues.     

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.     

Calcitonin receptors of human osteoclastoma

Osteoclast-rich cultures were prepared by disaggregation of osteoclastomas (giant cell tumour of bone) and settlement onto glass or plastic surfaces. Autoradiography using [125I]-salmon calcitonin ([125I]-sCT) revealed specific binding only to multinucleate giant cells (osteoclasts) and a minor population of mononuclear cells. [125I]-sCT competitive binding studies indicated a Kd of 5 x 10(-10) M and receptor number of approximately 1 million sites/osteoclast. sCT treatment resulted in a dose-dependent rise in cAMP (EC50 10(-10) M). Homogenates of an osteoclastoma also demonstrated specific binding of [125I]-sCT. Chemical cross-linking of a labelled synthetic sCT derivative. [125I]-[Arg11,18,Lys14]-sCT, using disuccinimidyl suberate, resulted in labelling of a receptor component of approximate Mr 85-90,000. The multinucleate giant cells (osteoclasts) of human osteoclastomas possess large number of CT receptors which exhibit the same binding kinetics and apparent Mr as those of other CT target cells.     

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.     

A reversible radioligand specific for the ETB receptor: [125I]Tyr13-Suc-[Glu9,Ala11,15]-endothelin-1(8- 21), [125I]IRL 1620.

Suc-[Glu9,Ala11,15]-endothelin(ET)-1(8-21), IRL 1620, is a linear ET-analog specific for the ET-isopeptide-nonselective ETB receptor. The radio-iodinated analog, [125I]IRL 1620, showed a single class of saturable binding to the ETB receptors in porcine lung membranes with a Kd of 18 pM and a Bmax of 930 fmol/mg protein, which are almost comparable to the values obtained with [125I]ET-3 (6 pM and 900 fmol/mg protein). In competitive binding assays with [125I]IRL 1620, unlabeled ET-1, ET-3, IRL 1620 and [monoiodo-Tyr13]-IRL 1620 showed almost identical displacement curves with Ki of 8 to 16 pM. However, [125I]IRL 1620 was dissociated from the binding sites by addition of an excess amount (100 nM) of any of these unlabeled peptides, each with the same t1/2 of 100 min. This was in marked contrast to [125I]ET-3 which was hardly dissociated from the binding sites.