TY - JOUR
T1 - Contribution of the transmembrane domain 6 of melanocortin-4 receptor to peptide [Pro5, dNal (2′)8]-γ-MSH selectivity
AU - Chen, Min
AU - Cai, Minying
AU - McPherson, David
AU - Hruby, Victor
AU - Harmon, Carroll M.
AU - Yang, Yingkui
N1 - Funding Information:
This work has been supported by NIH Grants R03 HD047312-01A1, 1 R03 HD058789-01 (Yang, Y-K) and U.S.P.H.S., N.I.H. grant DK 17420 (Hruby, V.J).
PY - 2009/1/1
Y1 - 2009/1/1
N2 - The melanocortin receptor (MCR) subtype family is a member of the GPCR superfamily and each of them has a different pharmacological profile regarding the relative potency of the endogenous and synthetic melanocortin peptides. Substitution of Trp with dNal (2′) in γ-MSH resulted in the loss of binding affinity and potency at hMC4R. However, the molecular mechanism of this ligand selectivity is unclear. In this study, we utilized chimeric receptors and site-directed mutagenesis approaches to investigate the molecular basis of MC4R responsible for peptide [Pro5, dNal (2′)8]-γ-MSH selectivity. Cassette substitutions of the second, third, fourth, fifth, and sixth TM of the human MC4R (hMC4R) with the homologous regions of hMC1R were constructed and the binding affinity of peptide [Pro5, dNal (2′)8]-γ-MSH at these chimeric receptors was evaluated. Our results indicate that the cassette substitutions of TM2, TM3, TM4 and TM5 of hMC4R with homologous regions of the hMC1R did not significantly increase peptide [Pro5, dNal (2′)8]-γ-MSH binding affinity and potency but substitution of the TM6 of the hMC4R with the same region of the hMC1R significantly enhances [Pro5, dNal (2′)8]-γ-MSH binding affinity and potency. Further site-directed mutagenesis study indicates that four amino acid residues, Phe267, Tyr268, Ile269 and Ser270, in TM6 of the hMC4R may play an important role in [Pro5, dNal (2′)-γ-MSH selective activity at MC4R.
AB - The melanocortin receptor (MCR) subtype family is a member of the GPCR superfamily and each of them has a different pharmacological profile regarding the relative potency of the endogenous and synthetic melanocortin peptides. Substitution of Trp with dNal (2′) in γ-MSH resulted in the loss of binding affinity and potency at hMC4R. However, the molecular mechanism of this ligand selectivity is unclear. In this study, we utilized chimeric receptors and site-directed mutagenesis approaches to investigate the molecular basis of MC4R responsible for peptide [Pro5, dNal (2′)8]-γ-MSH selectivity. Cassette substitutions of the second, third, fourth, fifth, and sixth TM of the human MC4R (hMC4R) with the homologous regions of hMC1R were constructed and the binding affinity of peptide [Pro5, dNal (2′)8]-γ-MSH at these chimeric receptors was evaluated. Our results indicate that the cassette substitutions of TM2, TM3, TM4 and TM5 of hMC4R with homologous regions of the hMC1R did not significantly increase peptide [Pro5, dNal (2′)8]-γ-MSH binding affinity and potency but substitution of the TM6 of the hMC4R with the same region of the hMC1R significantly enhances [Pro5, dNal (2′)8]-γ-MSH binding affinity and potency. Further site-directed mutagenesis study indicates that four amino acid residues, Phe267, Tyr268, Ile269 and Ser270, in TM6 of the hMC4R may play an important role in [Pro5, dNal (2′)-γ-MSH selective activity at MC4R.
KW - Agonist
KW - GPCR
KW - MC1R
KW - MC4R
KW - Obesity
KW - γ-MSH
UR - http://www.scopus.com/inward/record.url?scp=56649099031&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=56649099031&partnerID=8YFLogxK
U2 - 10.1016/j.bcp.2008.09.023
DO - 10.1016/j.bcp.2008.09.023
M3 - Article
C2 - 18930713
AN - SCOPUS:56649099031
SN - 0006-2952
VL - 77
SP - 114
EP - 124
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 1
ER -