The melanocortin (MC3) receptor from rat hypothalamus: photoaffinity labelling and binding of alanine-substituted alpha-MSH analogues: FEBS Lett

U. G. Sahm, M. A. Qarawi, G. W. Olivier, A. R. Ahmed, S. K. Branch, S. H. Moss, C. W. Pouton

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Membrane preparations of cells expressing the cloned rat hypothalamus melanocortin receptor, MC3, have been photoaffinity labelled using a radiolabelled photoreactive analogue of alpha-MSH, [125I-Tyr2,Nle4,D-Phe7,ATB-Lys11]alpha-MSH. SDS-PAGE followed by autoradiography showed a single band at 53-56 kDa for the native receptor or 35 kDa after deglycosylated with PNGase F, consistent with the predicted cDNA sequence. Receptor binding studies with alpha-MSH, gamma-MSH and [Nle4,D-Phe7]alpha-MSH established that alpha-MSH and gamma-MSH had similar affinities while [Nle4,D-Phe7]alpha-MSH bound 100 times more strongly. These results suggest that the receptor recognises the conserved 'core sequence' (-Met-Glu/Gly-His-Phe-Arg-Trp-) of MSH/ACTH peptides. The binding affinities of alanine-substituted analogues of alpha-MSH were determined to investigate the role of individual residues in ligand-receptor interactions. While in the terminal regions only the replacement of Tyr2 reduced the affinity of the peptide, replacement of Met4, Phe7, Arg8 and Trp9 within the peptide core led to a significant loss of affinity. Glu5 appeared unimportant for receptor recognition.
Original languageEnglish
Pages (from-to)29-32
Number of pages4
JournalFEBS Letters
Volume350
Publication statusPublished - 1994

Keywords

  • Affinity Labels Alanine/*metabolism Amino Acid Sequence Animals Cell Line, Transformed Humans Hypothalamus/*metabolism Iodine Radioisotopes Kinetics Molecular Sequence Data Photochemistry Rats Receptors, Corticotropin/*metabolism Receptors, Melanocortin Recombinant Proteins/metabolism alpha-MSH/*analogs & derivatives/metabolism

Cite this

Sahm, U. G. ; Qarawi, M. A. ; Olivier, G. W. ; Ahmed, A. R. ; Branch, S. K. ; Moss, S. H. ; Pouton, C. W. / The melanocortin (MC3) receptor from rat hypothalamus: photoaffinity labelling and binding of alanine-substituted alpha-MSH analogues : FEBS Lett. In: FEBS Letters. 1994 ; Vol. 350. pp. 29-32.
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title = "The melanocortin (MC3) receptor from rat hypothalamus: photoaffinity labelling and binding of alanine-substituted alpha-MSH analogues: FEBS Lett",
abstract = "Membrane preparations of cells expressing the cloned rat hypothalamus melanocortin receptor, MC3, have been photoaffinity labelled using a radiolabelled photoreactive analogue of alpha-MSH, [125I-Tyr2,Nle4,D-Phe7,ATB-Lys11]alpha-MSH. SDS-PAGE followed by autoradiography showed a single band at 53-56 kDa for the native receptor or 35 kDa after deglycosylated with PNGase F, consistent with the predicted cDNA sequence. Receptor binding studies with alpha-MSH, gamma-MSH and [Nle4,D-Phe7]alpha-MSH established that alpha-MSH and gamma-MSH had similar affinities while [Nle4,D-Phe7]alpha-MSH bound 100 times more strongly. These results suggest that the receptor recognises the conserved 'core sequence' (-Met-Glu/Gly-His-Phe-Arg-Trp-) of MSH/ACTH peptides. The binding affinities of alanine-substituted analogues of alpha-MSH were determined to investigate the role of individual residues in ligand-receptor interactions. While in the terminal regions only the replacement of Tyr2 reduced the affinity of the peptide, replacement of Met4, Phe7, Arg8 and Trp9 within the peptide core led to a significant loss of affinity. Glu5 appeared unimportant for receptor recognition.",
keywords = "Affinity Labels Alanine/*metabolism Amino Acid Sequence Animals Cell Line, Transformed Humans Hypothalamus/*metabolism Iodine Radioisotopes Kinetics Molecular Sequence Data Photochemistry Rats Receptors, Corticotropin/*metabolism Receptors, Melanocortin Recombinant Proteins/metabolism alpha-MSH/*analogs & derivatives/metabolism",
author = "Sahm, {U. G.} and Qarawi, {M. A.} and Olivier, {G. W.} and Ahmed, {A. R.} and Branch, {S. K.} and Moss, {S. H.} and Pouton, {C. W.}",
note = "M1 - 1 Sahm, U G Qarawi, M A Olivier, G W Ahmed, A R Branch, S K Moss, S H Pouton, C W eng Research Support, Non-U.S. Gov't NETHERLANDS 1994/08/15 FEBS Lett. 1994 Aug 15;350(1):29-32.",
year = "1994",
language = "English",
volume = "350",
pages = "29--32",
journal = "FEBS Letters",
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The melanocortin (MC3) receptor from rat hypothalamus: photoaffinity labelling and binding of alanine-substituted alpha-MSH analogues : FEBS Lett. / Sahm, U. G.; Qarawi, M. A.; Olivier, G. W.; Ahmed, A. R.; Branch, S. K.; Moss, S. H.; Pouton, C. W.

In: FEBS Letters, Vol. 350, 1994, p. 29-32.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - The melanocortin (MC3) receptor from rat hypothalamus: photoaffinity labelling and binding of alanine-substituted alpha-MSH analogues

T2 - FEBS Lett

AU - Sahm, U. G.

AU - Qarawi, M. A.

AU - Olivier, G. W.

AU - Ahmed, A. R.

AU - Branch, S. K.

AU - Moss, S. H.

AU - Pouton, C. W.

N1 - M1 - 1 Sahm, U G Qarawi, M A Olivier, G W Ahmed, A R Branch, S K Moss, S H Pouton, C W eng Research Support, Non-U.S. Gov't NETHERLANDS 1994/08/15 FEBS Lett. 1994 Aug 15;350(1):29-32.

PY - 1994

Y1 - 1994

N2 - Membrane preparations of cells expressing the cloned rat hypothalamus melanocortin receptor, MC3, have been photoaffinity labelled using a radiolabelled photoreactive analogue of alpha-MSH, [125I-Tyr2,Nle4,D-Phe7,ATB-Lys11]alpha-MSH. SDS-PAGE followed by autoradiography showed a single band at 53-56 kDa for the native receptor or 35 kDa after deglycosylated with PNGase F, consistent with the predicted cDNA sequence. Receptor binding studies with alpha-MSH, gamma-MSH and [Nle4,D-Phe7]alpha-MSH established that alpha-MSH and gamma-MSH had similar affinities while [Nle4,D-Phe7]alpha-MSH bound 100 times more strongly. These results suggest that the receptor recognises the conserved 'core sequence' (-Met-Glu/Gly-His-Phe-Arg-Trp-) of MSH/ACTH peptides. The binding affinities of alanine-substituted analogues of alpha-MSH were determined to investigate the role of individual residues in ligand-receptor interactions. While in the terminal regions only the replacement of Tyr2 reduced the affinity of the peptide, replacement of Met4, Phe7, Arg8 and Trp9 within the peptide core led to a significant loss of affinity. Glu5 appeared unimportant for receptor recognition.

AB - Membrane preparations of cells expressing the cloned rat hypothalamus melanocortin receptor, MC3, have been photoaffinity labelled using a radiolabelled photoreactive analogue of alpha-MSH, [125I-Tyr2,Nle4,D-Phe7,ATB-Lys11]alpha-MSH. SDS-PAGE followed by autoradiography showed a single band at 53-56 kDa for the native receptor or 35 kDa after deglycosylated with PNGase F, consistent with the predicted cDNA sequence. Receptor binding studies with alpha-MSH, gamma-MSH and [Nle4,D-Phe7]alpha-MSH established that alpha-MSH and gamma-MSH had similar affinities while [Nle4,D-Phe7]alpha-MSH bound 100 times more strongly. These results suggest that the receptor recognises the conserved 'core sequence' (-Met-Glu/Gly-His-Phe-Arg-Trp-) of MSH/ACTH peptides. The binding affinities of alanine-substituted analogues of alpha-MSH were determined to investigate the role of individual residues in ligand-receptor interactions. While in the terminal regions only the replacement of Tyr2 reduced the affinity of the peptide, replacement of Met4, Phe7, Arg8 and Trp9 within the peptide core led to a significant loss of affinity. Glu5 appeared unimportant for receptor recognition.

KW - Affinity Labels Alanine/metabolism Amino Acid Sequence Animals Cell Line, Transformed Humans Hypothalamus/metabolism Iodine Radioisotopes Kinetics Molecular Sequence Data Photochemistry Rats Receptors, Corticotropin/metabolism Receptors, Melanocortin Reco

M3 - Article

VL - 350

SP - 29

EP - 32

JO - FEBS Letters

JF - FEBS Letters

SN - 0014-5793

ER -