A method for mapping peptide receptors

Schwyzer, R.; Kriwaczek, V. M.; Wunderlin, R.

doi:10.1007/bf01047233

Cited by 9 publications

(17 citation statements)

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“…Pretreatment The present results are in complete agreement with our earlier results on fluorescence labeling of these cells using a water-soluble class of fluorescent melanotropic macromolecular reagents (6, 7) as well as with those of Schwyzer and coworkers, who developed conjugates of MSH with tobacco mosaic virus (8)(9)(10)(11)(12). Using the melanotropin fluorescein conjugate, we demonstrated that both melanotic as well as amelanotic human melanoma cells expressed membrane melanocortin receptors (6,7).…”

Section: Discussionsupporting

confidence: 92%

“…In this respect, these results are similar to electron microscopic visualization of internalized receptor-ligand complex for a glucagon-serum albumin preparation labeled with 17 nanometer gold particles (23). These results are consistent with those obtained earlier using fluorescent tobacco mosaic virus-MSH conjugates (8)(9)(10)(11)(12) and fluorescent polyvinyl alcohol-MSH conjugates (6, 7).…”

Section: Synthesis Of Peptide-conjugated Beadssupporting

confidence: 91%

“…This binding undoubtedly resulted from the multiplicity of the peptide ligand on these solid-phase probes, such that a single assembly of the bead-peptide conjugate is most likely able to interact simultaneously with more than one receptor on the cell membrane. This behavior has earlier been reported on interaction of tobacco mosaic virus-MSH conjugates with mouse melanoma cells (8)(9)(10)(11)(12). Similarly, certain classes of polyvalent antibodies that also are capable of establishing multiple interactions exhibit much higher binding affinities (22).…”

Section: Synthesis Of Peptide-conjugated Beadssupporting

confidence: 65%

“…The micro-and macrospheres in which the melanotropin analog was linked to the beads by a disulfide linkage (10) provided a control experiment to establish the specificity of the interactions between MSH beads and melanoma cells as described by Schwyzer and coworkers (8,10). Reducing agents such as DTT and 2-mercaptoethanol were able to reduce the S-S bond and therefore release the hormone analog.…”

Section: Disulfide Linkage Disruption (By 2-mercaptoethanol or Dtt)mentioning

confidence: 99%

“…These methods provide simple alternatives to the use of fluorescence techniques previously described by Schwyzer and coworkers (8)(9)(10)(11)(12) and recently by us (6,7). The specificity of these newer solid-phase reagents for MSH-binding sites is addressed and established.…”

mentioning

confidence: 99%

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Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors

Sharma

Jiang

Hadley

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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We developed two solid-phase reagent systems for microscopic visualization and characterization of melanocyte-stimulating hormone ( The search for a molecular marker for melanoma cancer cells is pivotal for the development of modalities for the early detection and management of melanoma. The identification of a specific universal antigen associated with melanoma has remained elusive. Usually, good antibody candidates specific for a particular melanoma specimen fail to recognize other tumor variants from different patients and sometimes even from the same individual (1, 2). The occurrence of melanotic as well as amelanotic tumors further illustrates the great degree of melanoma variance and the problem of unambiguous detection. A group of acidic cytosolic proteins, commonly referred to as S-100 proteins, have been claimed to be a useful marker for melanoma (3, 4). An immunoassay based on this protein has been used to screen formalin fixed melanoma tumors in vitro, stressing its importance as a good diagnostic tool for melanoma, especially in case of metastatic lymph node melanoma. Unfortunately, a large number of normal tissues also contain S-100 proteins in substantial amounts (5), making it a rather complex issue in distinguishing the truly positive from the truly negative specimens. Furthermore, the cytosolic localization of these proteins suggests that antibody-directed targeting of modalities of therapeutic relevance might not be feasible. For this purpose, the identification of specific cell membrane markers is better suited because of their rather facile accessibility by cell-specific targeting agents.We recently described the development of melanotropin conjugates that could detect the presence of melanotropin receptors on a number of mouse and human melanoma cell lines, melanotic as well as amelanotic, that we investigated (6, 7). These conjugates were composed of multiple copies of both a potent melanotropin analog and a fluorophore that were conjugated to a biologically inert macromolecule in a pendant fashion. The multivalency of the ligand molecule in these conjugates afforded specific binding between the conjugate and the melanotropin receptors, presumably by establishing simultaneous interactions with a number of melanocytestimulating hormone (MSH) receptors on the cell membrane. In addition, these macromolecular composites also allowed visualization of certain phenomena, like capping and internalization, that are associated with certain ligand-receptor binding.Here we describe another class of multivalent melanotropic ligands that can detect the presence or absence of specific MSH binding sites on melanoma cells in a very simple and straightforward manner. These methods provide simple alternatives to the use of fluorescence techniques previously described by and recently by us (6, 7). The specificity of these newer solid-phase reagents for MSH-binding sites is addressed and established. MATERIALS AND METHODSN, N-Dimethylaminopyridine was obtained from Aldrich. DTT, mercaptoethanol, 4-( p-...

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Section: Discussionsupporting

confidence: 92%

Section: Synthesis Of Peptide-conjugated Beadssupporting

confidence: 91%

Section: Synthesis Of Peptide-conjugated Beadssupporting

confidence: 65%

Section: Disulfide Linkage Disruption (By 2-mercaptoethanol or Dtt)mentioning

confidence: 99%

mentioning

confidence: 99%

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Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors

Sharma

Jiang

Hadley

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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Tobacco mosaic virus as a carrier for small molecules: Artificial receptor antibodies and superhormones

Schwyzer

Kriwaczek

1981

Biopolymers

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SynopsisPeptide agonists covalently attached to tobacco mosaic virus exhibit such unusual properties as superpotency, superaffinity, enhanced resistance towards enzymic degradation, and prolonged action at the target cell. These properties can be exploited for the isolation by density-gradient centrifugation of membrane vesicles bearing specific receptors for the peptides and for radioactive and fluorescent labeling of cell-surface receptors. Our observations can be explained by cooperative-affinity phenomena caused by the deployment in space of the agonist molecules. DEPLOYMENT OF AGONIST MOLECULES AS A FACTOR IN BIOLOGICAL ACTIVITYSince early work on the conformation of cyclic peptides and on the organization of information in linear peptide agonists (for reviews, see Refs. 1-3), it has become increasingly clear that there exist causal relationships between the amino acid sequences of peptides, their conformational possibilities, their interactions with receptors, and their biological activity. In this review, we would like to point out the importance of another factor governing receptor interactions and biological activity, namely the deployment in space of groups of individual agonist molecules. Cooperative AffinityThe strong affinity of antibodies for membrane-bound antigens has been explained by the simultaneous binding of the two recognition sites on the antibody molecule to two antigen molecules connected through the membrane.4.5 Cooperative affinity effects of this type should prevail in any situation in which ligand and acceptor each possess more than one mutual recognition or binding site, provided that these sites are deployed in such a manner that simultaneous interaction is stereochemically feasible. A classical example of almost perfect complementarity is the DNA double helix, in which one single strand can be regarded as the ligand and the other as the acceptor molecule, both containing favorably deployed binding sites: the purine and pyrimidine bases.

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Melanotropin Receptors I. Synthesis and Biological Activity of N^α‐(5‐Bromovaleryl)‐N^α‐deacetyl‐α‐melanotropin

Wunderlin

Minakakis

Tun‐Kyi

et al. 1985

Helvetica Chimica Acta

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Chemical synthesis and biological activities of a new a-melanotropin derivative are described. Nu-(5-Bro-movalery1)-Na-deacetyl-a-melanotropin contains the 5-bromopentanoyl group as a chemical 'handle' in place of the acetyl group of the natural hormone. The synthesis involved a new protected intermediate which allowed the selective deprotection of either the N" or N' amino group. The title compound reacted with sodium thiosulfate to give N"-deacetyl-Na-(5-(sulfothio)valeryl)-a-melanotropin, a key intermediate for the preparation of tobaccomosaic virus/a-melanotropin disulfide conjugates. As a basis for the study of the conjugates, biological activities of the title compound on Cloudman S-YI mouse melanoma cell cultures (tyrosinase stimulation, binding, and cyclic AMP accumulation) were determined. They proved to be quite similar to the corresponding a-melanotropin activities. Differences in bindings may be explained by stronger hydrophobic interaction of the new derivative with the lipid phase of the target cell membranes. ') Parts of this report have appeared as a thesis [I]. Nomenclature and abbreviations [2]. Additional abbreviations: MSH = rnelanophore stimulating hormone (melanotropin), DCC = N,N-dicyclohexylcarbodiimide, DCU = N,N'-dicyclohexylurea, DMF = dimethylformamide, HOBt = 1-hydroxybenzotriazole, Boc = tert-butoxycarbonyl, OBu' = tert-butoxy, Msoc = 2-(methylsulfonyl)ethoxycarbonyl, Np = 4-nitrophenyl, Z = benzyloxycarbonyl, TFA = trifluoroacetic acid. Chiral amino acids are in their L-configuration. Culture media see E.xperimentu1. Synthesis of N"-(5-Bromovaleryl)-N"-deacetyl-a-melanotropin (10).-Two approaches are illustrated in the Scheme. One (steps 11+12-+10) was based on an earlier synthesis of a-MSH [7] wherein the Lys (Msoc)-11 protection is removed by alcaline /I-elimination in the last step. This worked here for the relatively stable 5-bromovaleryl derivative. However, for more reactive handles, such as bromoacetyl or maleimido groups [5] [8], deblocking in the final stage must be effected by mild acid treatment. We, therefore, reversed the type of protection, using Msoc for the IT-atom and protecting groups derived from t-butyl alcohol for the side chains [9]. The key intermediates 7 and 8 can serve a wider range of handles than 11.Suitable educts for the second approach (steps l+lO) were the Boc-tetrapeptide methyl ester 1 [lo] and the Z-protected nonapeptide 5 [ll]. The former was saponified to the free acid 2 which was treated with TFA to remove the N-terminal protection. The crystalline tetrapeptide salt 3. TFA was reacted with 4-nitrophenyl 2-(methyl-sulfony1)ethyl carbonate [7] [ 121 to yield crystalline, analytically pure Msoc-tetrapeptide 4. Its isolation was rather difficult because of a pronounced solubility in H,O and organic solvents.Compound 4 was condensed with the mixed HOBt.HC1 salt 6 of partially protected nonapeptide amide (produced from 5 by catalytic hydrogenation in the presence of HOBt), using the DCC/HOBt method without base. This procedure is known to minimize sid...

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A method for mapping peptide receptors

Cited by 9 publications

References 6 publications

Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors

Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors

Tobacco mosaic virus as a carrier for small molecules: Artificial receptor antibodies and superhormones

Melanotropin Receptors I. Synthesis and Biological Activity of N^α‐(5‐Bromovaleryl)‐N^α‐deacetyl‐α‐melanotropin

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A method for mapping peptide receptors

Cited by 9 publications

References 6 publications

Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors

Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors

Tobacco mosaic virus as a carrier for small molecules: Artificial receptor antibodies and superhormones

Melanotropin Receptors I. Synthesis and Biological Activity of Nα‐(5‐Bromovaleryl)‐Nα‐deacetyl‐α‐melanotropin

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Melanotropin Receptors I. Synthesis and Biological Activity of N^α‐(5‐Bromovaleryl)‐N^α‐deacetyl‐α‐melanotropin