Hexapeptides that interfere with HIV-1 fusion peptide activity in liposomes block GP41-mediated membrane fusion

Upon receptor-mediated activation, the gp41 hydrophobic, conserved fusion peptide inserts into the target membrane and promotes the kind of perturbations required for the progression of the HIV-cell fusion reaction. Using a synthetic combinatorial library we have identified all d-amino acid hexapeptide sequences that inhibited the fusion peptide capacity of perturbing model membranes. Two hexapeptides that effectively inhibited the fusion peptide in these systems were subsequently shown to inhibit cell-cell fusion promoted by gp41 expressed at cell surfaces. These observations might be of importance for understanding the mechanisms underlying fusion peptide activity and suggest new strategies for screening compounds that target these viral sequences.     

Predicted calmodulin-binding sequence in the gamma subunit of phosphorylase b kinase

A basic, amphiphilic alpha helix is a structural feature common to a variety of inhibitors of calmodulin and to the calmodulin-binding domains of myosin light chain kinases. To aid in recognizing this structural feature in sequences of peptides and proteins we have developed a computer algorithm which searches for sequences of appropriate length, hydrophobicity, helical hydrophobic moment, and charge to be considered as potential calmodulin-binding sequences. Such sequences occurred infrequently in proteins of known crystal structure. This algorithm was used to find the most likely site in the catalytic (gamma) subunit of phosphorylase b kinase for interaction with calmodulin (the delta subunit). A peptide corresponding to this site (residues 341-361 of the gamma subunit) was synthesized and found to bind calmodulin with approximately an 11 nM dissociation constant. A variant of this peptide in which an aspartic acid at position 7 in its sequence (347 of the gamma subunit) was replaced with an asparagine was found to bind calmodulin with approximately a 3 nM dissociation constant.     

Synthesis,characterization, and biological activity of poly(arginine)‐derived cancer‐targeting peptides in HepG2 liver cancer cells

The solid‐phase synthesis, structural characterization, and biological evaluation of a small library of cancer‐targeting peptides have been determined in HepG2 hepatoblastoma cells. These peptides are based on the highly specific Pep42 motif, which has been shown to target the glucose‐regulated protein 78 receptors overexpressed and exclusively localized on the cell surface of tumors. In this study, Pep42 was designed to contain varying lengths (3–12) of poly(arginine) sequences to assess their influence on peptide structure and biology. Peptides were effectively synthesized by 9‐fluorenylmethoxycarbonyl‐based solid‐phase peptide synthesis, in which the use of a poly(ethylene glycol) resin provided good yields (14–46%) and crude purities >95% as analyzed by liquid chromatography–mass spectrometry. Peptide structure and biophysical properties were investigated using circular dichroism spectroscopy. Interestingly, peptides displayed secondary structures that were contingent on solvent and length of the poly(arginine) sequences. Peptides exhibited helical and turn conformations, while retaining significant thermal stability. Structure–activity relationship studies conducted by flow cytometry and confocal microscopy revealed that the poly(arginine) derived Pep42 sequences maintained glucose‐regulated protein 78 binding on HepG2 cells while exhibiting cell translocation activity that was contingent on the length of the poly(arginine) strand. In single dose (0.15 mM) and dose‐response (0–1.5 mM) cell viability assays, peptides were found to be nontoxic in human HepG2 liver cancer cells, illustrating their potential as safe cancer‐targeting delivery agents. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Multiple biological activities for two peptides derived from the nerve growth factor precursor

ProNGF can be cleaved proteolytically at dibasic residues and liberates two other peptides beside NGF, LIP1 a 29 amino acid (aa) peptide and LIP2 a 38 aa peptide. These peptides were found present in the rat intestine and shown to induce rapid phosphorylation of the Trk receptor in cell lines. The present study describes several novel biological properties for these peptides. They exert an anti-proliferative effect on the mitogenic activity of estrogen and IGF in MCF-7 cells. They protect against in vivo induction of excitotoxic lesions by the glutamatergic analogue ibotenate injected into the developing mouse brain and against in vitro NMDA-induced cell death in primary neuronal cultures. They bind to murine microglial cells and induce phosphorylation of Akt. These results suggest a role for LIP1 and LIP2 in cell survival.     

Amino acid sequences of alpha-helical segments from S-carboxymethylkerateine-A. Tryptic and chymotryptic peptides from a type-II segment.

1. Amino acid-sequence studies were done on a peptide of mol.wt. approx. 12500 that was isolated from the highly helical fragments obtained by partial chymotryptic digestion of the low-sulphur proteins (S-carboxymethylkerateine-A) from wool. 2. The peptides obtained by tryptic and chymotryptic digestion of this large peptide were separated by ion-exchange chromatography on DEAE-cellulose at pH8.5 with an (NH4)(2)CO(3) concentration gradient and, where necessary, purified further by paper electrophoresis. 3. Determination of the sequences of many of these peptides showed that a high proportion of the cationic residues occurs in pairs. 4. Although two of the four S-carboxymethylcysteine residues are located in what appears to be a non-helical region near the N-terminus the other two S-carboxymethylcysteine residues occur in or near sequences suggesting a helical conformation. 5. Some peptides were obtained, in low yields, that appeared to be homologues of more major ones. These suggest either homologies in the helical portions of the low-sulphur proteins or the presence of closely related amino acid sequences in helical regions of completely different origins. 6. A partial sequence of the complete peptide is proposed.     

Identification of a Ca2+/calmodulin-binding domain within the carboxyl-terminus of the angiotensin II (AT1A) receptor.

To identify regulators of the type 1A angiotensin II receptor (AT1A), we investigated the interaction of cellular proteins with a fusion protein containing the rat AT1A receptor carboxyl-terminus. An approximately 20 kDa cytoplasmic protein interacted with the fusion protein in a Ca2+-dependent manner and was identified as calmodulin. A control peptide with high affinity for Ca2+/calmodulin and a peptide corresponding to a membrane proximal portion of the AT1A receptor carboxyl-terminus with analogy to known calmodulin-binding sequences were synthesised and tested for calmodulin-binding. Using in vitro binding assays combined with gel shift analysis, we demonstrated the formation of complexes between calmodulin and both peptides, which were Ca2+-dependent and of 1:1 stoichiometry. Affinity gels produced from these peptides also purified calmodulin from cell extracts. These results suggest a novel feedback regulation of the AT1A receptor by Ca2+/calmodulin and identify the membrane proximal region of the carboxyl-terminus as a focal point for interactions important for AT1A receptor function.     

Phage matrix for isolation of glioma cell membrane proteins

Cell-binding ligands for RG2 rat glioma were identified in our recent study from a library of peptides that are displayed as fusion molecules on phage particles. Here, one of the phage clones was used to affinity purify those cell membrane components to which the displayed peptides bind. This phage clone, displaying the ELRGDSLP peptide, was shown to recognize glioma cells specifically in comparison to control phage-expressing peptides of either similar or irrelevant sequences. Blocking experiments with synthetic RGDS peptide demonstrated that the phage-glioma cell recognition occurs via the RGD motif known to be present in many integrin-binding proteins. To form an affinity matrix that would bind to glioma cell membrane molecules, ELRGDSLP phage particles were cross-linked using dextran polymer. Whole cell lysate from RG2 rat glioma cells was passed through the matrix, resulting in the isolation of cell membrane components having strong affinity to the peptides on phage and molecules associated with those components. One of the isolated proteins was found to be CD44s, a cell surface adhesion molecule involved in glioma cell invasion and migration, which likely formed a complex with an RGD-binding integrin. Cell membrane proteins isolated with this innovative approach could be used for the design of cell-specific anticancer treatments.     

Stabilization of an alpha-helical conformation in an isolated hexapeptide inhibitor of calmodulin.

The conformational properties of two hexapeptides, Ac-LWRILW-NH(2) and its D-amino acid counterpart Ac-lwrilw-NH(2), identified as calmodulin inhibitors using mixture-based synthetic combinatorial library approaches, have been characterised by NMR and CD spectroscopy. The peptides fold into an alpha-helical conformation in aqueous solution. The observed short- and medium-range nuclear Overhauser effects were consistent with the formation of an alpha-helical structure and a reasonably well-defined set of structures was obtained by using restraints from the NMR data in simulated annealing calculations. Analysis of glycine-substitution analogues demonstrated that all the amino acids that make up the peptide sequence are important for the stabilization of the alpha-helical conformation. The results suggest that a well-defined set of interactions is indispensable to allow alpha-helix formation in this short hexapeptide.     

Identification of residues necessary for clonally specific recognition of a cytotoxic T cell determinant.

The residues in an influenza nucleoprotein (NP) cytotoxic T cell determinant necessary for cytotoxic T cell (CTL) recognition, were identified by assaying the ability of hybrid peptides to sensitize a target cell to lysis. The hybrid peptides were formed by substituting amino acids from one determinant (influenza NP 147-158) for the corresponding residues of a second peptide (HLA CW3 171-182) capable of binding to a common class I protein (H-2Kd). Six amino acids resulted in partial recognition; however, the presence of a seventh improved the potency of the peptide. Five of the six amino acids were shown to be required for recognition. The spacing of the six amino acids was consistent with the peptide adopting a helical conformation when bound. The importance of each amino acid in CTL recognition and binding to the restriction element was investigated further by assaying the ability of peptides containing point substitutions either to sensitize target cells or to compete with the natural NP sequence for recognition by CTL. The T cell response was much more sensitive to substitution than the ability of the peptide to bind the restriction element. Collectively the separate strategies identified an approximate conformation and orientation of the peptide when part of the complex and permitted a potential location in the MHC binding site to be identified. The model provides a rationalization for analogues which have previously been shown to exhibit greater affinity for the class I molecule and suggests that the binding site in major histocompatibility complex (MHC) class I molecules might have greater steric constraints that the corresponding area of class II proteins.     

Cell adhesive peptide screening of the mouse laminin α1 chain G domain

Cell adhesive peptides have been widely applied for therapeutic drugs, drug delivery systems, and biomaterials. Previously, we identified various cell adhesive sequences in the G domains of four laminin α chains (α2-α5) by the systematic soluble peptide screening. We also identified five cell-binding sequences in the laminin α1 chain G domain using synthetic peptide-polystyrene beads. Here, we re-screened cell adhesive peptides in the laminin α1 chain G domain by the systematic soluble peptides screening. The 110 soluble peptides were evaluated for their cell adhesive activities using human fibrosarcoma HT1080 cells and human dermal fibroblasts. Fourteen peptides were newly identified as a cell adhesive. Additionally, four peptides (AG22: SSFHFDGSGYAM, AG42: TFDLLRNSYGVRK, AG76: HQNQMDYATLQLQ, AG86: LGGLPSHYRARNI) promoted integrin-mediated cell adhesion. Further, neurite outgrowth activity with rat pheochromocytoma PC12 cells was evaluated and two peptides (AG20: SIGLWNYIEREGK, AG26: SPNGLLFYLASNG) were newly identified for neurite outgrowth activity. These results suggested that the systematic soluble peptides screening approach is an accurate and powerful strategy for finding biologically active sequences. The active sequences newly identified here could be involved in the biological functions of this domain. The active peptides are useful for evaluating molecular mechanisms of laminin-receptor interactions and for developing cell adhesive biomaterials.     

Molecular cloning of a human co-beta-glucosidase cDNA: evidence that four sphingolipid hydrolase activator proteins are encoded by single genes in humans and rats

Authentic cDNAs encoding the activator protein for acid beta-glucosidase (EC3.2.1.45), co-beta-glucosidase, were cloned from the pCD and lambda gt11 human cDNA libraries. Initial screening with oligonucleotide mixtures encoding amino acid sequences of co-beta-glucosidase identified partial cDNAs which were used to obtain a potentially full-length cDNA from the lambda gt11 library. This clone (2767 bp), EGTISI, contained 5' (38 bp) and 3' (1157 bp) noncoding sequences, a translation initiation site, and an open reading frame encoding 524 amino acids which included a typical hydrophobic signal sequence (16 amino acids). Computer analyses identified three regions of high similarity to co-beta-glucosidase encoded by tandem sequences in EGTISI. Searches revealed that two of these regions encoded peptides of known function; SAP1 (sphingolipid activator protein 1) and protein C (a new sphingolipid activator protein) were encoded by EGTISI sequences 5' and 3', respectively, to those for co-beta-glucosidase. The third region of similarity, encoding a theoretical peptide (undefined function), was located most 5' in the cDNA. EGTISI and its encoded polypeptide had high similarity (77% nucleotide identity and about 80% amino acid similarity) to a rat Sertoli cell cDNA and its encoded sulfated glycoprotein-1. These results indicate that a single highly conserved gene encodes the precursor for four potential sphingolipid activator proteins in rat and man.     

Transient vesicle leakage initiated by a synthetic apoptotic peptide derived from the death domain of neurotrophin receptor, p75NTR.

Peptides that induce apoptosis have potential as anticancer therapeutics. The design of safe, effective cancer therapeutic peptides requires characterization of the physical and chemical properties that influence activation of cell death in neoplastic cells. NTR365 is a synthetic pro-apoptotic peptide with an amino acid sequence derived from the death domain of p75(NTR). These studies were initiated to identify a potential mechanism for the apoptotic activity of NTR365 identified by Rabizadeh et al. We examined the interactions of this synthetic pro-apoptotic peptide with phospholipid vesicles. Fluorescence experiments demonstrate that the peptide induces leakage from large unilamellar vesicles. Leakage activity is transient and dependent on the presence of anionic lipid in the vesicles. Circular dichroism studies show that the NTR365 adopts a different conformation and may have altered vesicle affinity under conditions conducive to leakage. The active conformation of NTR365 differs from that of the NMR derived conformation. A related peptide with a single substitution is not apoptotically active, does not form a helical structure in the presence of vesicles and does not induce appreciable vesicle leakage under the same conditions as NTR365. These studies suggest that the demonstrated apoptotic activity of a closely related NTR364 peptide is linked to disruption of a membrane barrier and to the ability of the peptide to form a helical structure.     

Structural model of HLA-DR1 restricted T cell antigen recognition

Two human helper T cell determinants in influenza have been identified, one in the hemagglutinin and the other in the matrix protein (M1). Both were shown to be DR1 restricted by using transfected L cells to present antigen. Comparison of the sequences of the two peptides revealed a similar pattern that could account for their DR1 specificity if the peptides adopt a helical conformation. The model was supported by the demonstration that hybrid peptides, composed of the amino acids that interact with DR1 from one determinant and the residues that interact with the T cell receptor from the other, were recognized by each clone. The generality of the motif was confirmed by the finding that DR1 individuals respond to a ragweed peptide containing the defined pattern.     

Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. I. Myohemerythrin.

In an attempt to delineate potential folding initiation sites for different protein structural motifs, we have synthesized series of peptides that span the entire length of the polypeptide chain of two proteins, and examined their conformational preferences in aqueous solution using proton nuclear magnetic resonance and circular dichroism spectroscopy. We describe here the behavior of peptides derived from a simple four-helix bundle protein, myohemerythrin. The peptides correspond to the sequences of the four long helices (the A, B, C and D helices), the N- and C-terminal loops and the connecting sequences between the helices. The peptides corresponding to the helices of the folded protein all exhibit preferences for helix-like conformations in solution. The conformational ensembles of the A- and D-helix peptides contain ordered helical forms, as shown by extensive series of medium-range nuclear Overhauser effect connectivities, while the B- and C-helix peptides exhibit conformational preferences for nascent helix. All four peptides adopt ordered helical conformations in mixtures of trifluoroethanol and water. The terminal and interconnecting loop peptides also appear to contain appreciable populations of conformers with backbone phi and psi angles in the alpha-region and include highly populated hydrophobic cluster and/or turn conformations in some cases. Trifluoroethanol is unable to drive these peptides towards helical conformations. Overall, the peptide fragments of myohemerythrin have a marked preference towards secondary structure formation in aqueous solution. In contrast, peptide fragments derived from the beta-sandwich protein plastocyanin are relatively devoid of secondary structure in aqueous solution (see accompanying paper). These results suggest that the two different protein structural motifs may require different propensities for formation of local elements of secondary structure to initiate folding, and that there is a prepartitioning of conformational space determined by the local amino acid sequence that is different for the helical and beta-sandwich structural motifs.     

hCXCR1 and hCXCR2 antagonists derived from combinatorial peptide libraries

The human CXCL8 plays important roles in inflammation by activation of neutrophils through the hCXCR1 and hCXCR2 receptors. The role of hCXCR1 and hCXCR2 in the pathogenesis of inflammatory responses has encouraged the development of antagonists of these receptors. In this study, we used phage display peptide libraries to identify peptides antagonists that block the interactions between hCXCL8 and hCXCR1/2. Two linear hexapeptides (MSRAKE and CAKELR) and two disulfide-constrained hexapeptides (CLRSGRFC and CLPWKENC) were recovered by panning phage libraries on hCXCR1- and hCXCR2-transfected murine pre-B cells after specific elution with hCXCL8. Sequence analysis revealed homology between the linear hexapeptides and the N-terminal domain (1-SAKELR-6), whereas the constrained peptides are composed of non-contiguous amino acids mimicking spatial structure on the surface of folded C-terminal portion of hCXCL8 (50-CLDPKENWVQRVVEKFLKRAENS-72). The synthetic linear and structurally constrained peptides competed for (125)I-hCXCL8 binding to hCXCR1 and hCXCR2 (IC(50) comprised between 10 and 100μM). Furthermore, they inhibited the intracellular calcium flux and the migration of hCXCR1/hCXCR2 transfectants; and desensitized hCXCR1 and hCXCR2 receptors on neutrophils, reducing their chemotactic responses induced by ELR-CXC chemokines (hCXCL8, hCXCL1, hCXCL2, hCXCL3, and hCXCL5). To better characterize the residues required for hCXCL8 binding, we identified three linear peptides MLRQTR, HASILP and KKEPWI specific to hCXCL8. These peptides similarly displaced the binding of (125)I-hCXCL8 to hCXCR1 (IC(50) ranging from 8.5 to 10μM) in a dose-dependent manner, inhibited hCXCL8 induced increases in the intracellular calcium, and migration of hCXCR1- and hCXCR2-transfected cells. The identified peptides could be used as antagonists of hCXCL8-induced activities related to its interaction with hCXCR1 and hCXCR2 receptors and may help in the design of new anti-inflammatory therapeutic molecules.     

wrwyrggrywrw is a single-chain functional analog of the Holliday junction-binding homodimer, (wrwycr)2

DNA repair pathways in bacteria that use homologous recombination involve the formation and subsequent resolution of Holliday junction (HJ) intermediates. We have previously identified several hexameric peptides that bind to HJs and interfere with HJ processing enzymes in vitro. The peptide WRWYCR and its D-amino acid stereoisomer wrwycr, are potent antibacterial agents. These hexapeptides must form homodimers in order to interact stably with HJs, and inhibit bacterial growth, and this represents a potential limitation. Herein we describe a disulfide bond-independent inhibitor, WRWYRGGRYWRW and its D-stereoisomer wrwyrggrywrw. We have characterized these single-chain, linear analogs of the hexapeptides, and show that in addition to effectively binding to HJs, and inhibiting the activity of DNA repair enzymes that process HJs, they have equal or greater potency against Gram-positive and Gram-negative bacterial growth. The analogs were also shown to cause DNA damage in bacteria, and disrupt the integrity of the bacterial cytoplasmic membrane. Finally, we found that they have little toxicity toward several eukaryotic cell types at concentrations needed to inhibit bacterial growth.     

Identification of novel hexapeptides bioactive against phytopathogenic fungi through screening of a synthetic peptide combinatorial library

The purpose of the present study was to improve the antifungal activity against selected phytopathogenic fungi of the previously identified hexapeptide PAF19. We describe some properties of a set of novel synthetic hexapeptides whose D-amino acid sequences were obtained through screening of a synthetic peptide combinatorial library in a positional scanning format. As a result of the screening, 12 putative bioactive peptides were identified, synthesized, and assayed. The peptides PAF26 (Ac-rkkwfw-NH(2)), PAF32 (Ac-rkwhfw-NH(2)), and PAF34 (Ac-rkwlfw-NH(2)) showed stronger activity than PAF19 against isolates of Penicillium digitatum, Penicillium italicum, and Botrytis cinerea. PAF26 and PAF32, but not PAF34, were also active against Fusarium oxysporum. Penicillium expansum was less susceptible to all four PAF peptides, and only PAF34 showed weak activity against it. Assays were also conducted on nontarget organisms, and PAF26 and PAF32 showed much-reduced toxicity to Escherichia coli and Saccharomyces cerevisiae, demonstrating selectivity towards certain filamentous fungi. Thus, the data showed distinct activity profiles for peptides differentiated by just one or two residue substitutions. Our conclusion from this observation is that a specificity factor is involved in the activity of these short peptides. Furthermore, PAF26 and PAF32 displayed activities against P. digitatum, P. italicum, and B. cinerea similar to that of the hemolytic 26-amino acid melittin, but they did not show the high toxicity of melittin towards bacteria and yeasts. The four peptides acted additively, with no synergistic interactions among them, and PAF26 was shown to have improved activity over PAF19 in in vivo orange fruit decay experiments.     

Formation of cyclic imide-like structures upon the treatment of calmodulin and a calmodulin peptide with heat

Protein cyclic imide is the putative intermediate in the formation of sites of carboxyl-methylation in eukaryotic proteins. Conditions known to induce the formation of a cyclic imide in model peptides have been applied to a protein, calmodulin. Heating of calmodulin in the dry state at 100 degrees C for 24 h after lyophilization from a pH 2.0 or pH 6.0 solution produces derivatives with altered chromatographic properties in anion-exchange HPLC. At pH 6.0, complete activity of calmodulin was retained. Analysis with Fourier transform infrared (FTIR)-photoacoustic spectroscopy demonstrated the presence of a new structure in the calmodulin molecule consistent with modification of carboxylic acid groups. The conversion of calmodulin is dependent upon the absence of Ca2+ (the presence of 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid). A peptide analogous to the calcium binding regions of calmodulin, Asp-Lys-Asp-Gly-Asn-Gly-Thr-Ile-Thr-Thr-Lys-Glu, is also converted, upon heating, to chromatographically different forms in reversed-phase chromatography. This process is also dependent upon the absence of calcium. Sequence analysis of the peptide derivatives reveals a second amino terminus, implicating peptide bond hydrolysis in the product. A dipeptide, Asp-Gly, known to form a cyclic imide structure under similar conditions is also hydrolyzed during sequence analysis consistent with cleavage occurring at the position of the cyclic imide structure. Asp3 is suggested to be the site of cyclic imide formation in the calmodulin peptide. The presence of a cyclic imide structure is also confirmed by the application of FTIR-photoacoustic spectroscopy. These data suggest that cyclic imide formation in calmodulin has been induced, possibly at one, or more, of the calcium binding loops of the protein. These modification reactions may provide a basis for future investigations of cyclic imide formation in proteins.     

Inhibition of normal human natural killer cell activity by human immunodeficiency virus synthetic transmembrane peptides

The inhibitory effect on normal natural killer (NK) cell activity of two synthetic peptides corresponding to amino acid sequences 735-752 and 846-860, respectively, as deduced from the amino acid sequences of HTLV-IIIB gp160, was assessed. Sequences 735-752 and 846-860 correspond to regions located within the HIV transmembrane gp41, the carboxy terminus of HIV gp160. These two synthetic peptides have been shown previously to suppress the mitogen- and alloantigen-induced normal human lymphocyte blastogenic responses. Peptides 735-752 and 846-860 conjugated to protein carriers exerted a significant inhibition on the normal NK cell activity assayed against K562 tumor target cells in an in vitro 51Cr-release cytoltoxicity assay. At variance, control peptides similarly conjugated had no effect on NK activity. Addition of exogenous recombinant human interleukin-2 (IL-2) resulted in a partial restoration of the suppression of NK cell activity exerted by both peptides. Binding experiments indicated that peptides 735-752 and 846-860 did not affect the formation of effector cell-target cell conjugates, suggesting inhibitory effect(s) subsequent to the formation of the lytic complex as one potential mechanism of the observed NK suppression. These results suggest that peptides 735-752 and 846-860 homologous to sequences within the HIV transmembrane gp41 may play an important role in the pathogenesis of the defective NK cell activity observed in patients with acquired immunodeficiency syndrome (AIDS).     

A synthetic analog of the calmodulin-binding domain of myosin light chain kinase inhibits melanotropin receptor function and activation of adenylate cyclase

In this study a synthetic analog of the calmodulin-binding domain of myosin light chain kinase, a 17-amino-acid peptide (M5) was used to examine the possible role of calmodulin in melanotropin receptor function. Binding of beta-melanocyte-stimulating hormone to its membrane receptor and subsequent stimulation of adenylate cyclase (AC) were found to be specifically inhibited by M5 in a dose-dependent and noncompetitive manner, both in intact M2R melanoma cells and in a plasma membrane preparation derived thereof. Half-maximal inhibition of both hormone binding and melanotropin-sensitive AC activity was shown to occur at approximately 1 microM M5. In contrast, stimulation of AC by prostaglandin E1, guanosine 5'-O-(3-thio)triphosphate, forskolin, and unstimulated enzyme activity were unaffected by the presence of M5, under the same assay conditions. Furthermore, addition of a molar excess of calmodulin to the AC assay completely abolished the inhibitory effects of the peptide on melanotropin-stimulated AC activity. Other peptides of similar size, which bind to calmodulin with low affinity (e.g. glucagon, somatostatin, and vasoactive intestinal peptide), were shown to be totally ineffective in inhibiting melanotropin-sensitive AC. These findings, along with those shown previously for other antagonists of calmodulin, suggest a role for an M5-binding protein, as of yet unidentified, involved in the regulation of the melanotropin receptor function.