Modulation of constitutive activity and signaling bias of the ghrelin receptor by conformational constraint in the second extracellular loop
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Modulation of constitutive activity and signaling bias of the ghrelin receptor by conformational constraint in the second extracellular loop. / Mokrosinski, Jacek; Frimurer, Thomas M; Sivertsen, Bjoern; Schwartz, Thue W; Holst, Birgitte.
In: Journal of Biological Chemistry, Vol. 287, 09.2012, p. 33488-33502.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - Modulation of constitutive activity and signaling bias of the ghrelin receptor by conformational constraint in the second extracellular loop
AU - Mokrosinski, Jacek
AU - Frimurer, Thomas M
AU - Sivertsen, Bjoern
AU - Schwartz, Thue W
AU - Holst, Birgitte
PY - 2012/9
Y1 - 2012/9
N2 - Based on a rare, natural Glu for Ala204(C+6) variant located six residues after the conserved Cys residue in extracellular loop 2 (ECL2b) associated with selective elimination of the high constitutive signaling of the ghrelin receptor, this loop was subjected to a detailed structure functional analysis. Introduction of Glu in different positions demonstrated that although the constitutive signaling was partly reduced when introduced in position 205(C+7) it was only totally eliminated in position 204(C+6). No charge-charge interaction partner could be identified for Glu(C+6) variant despite mutational analysis of a number of potential partners in the extracellular loops and outer parts of the transmembrane segments. Systematic probing of position 204(C+6) with amino acid residues of different physicochemical properties indicated that a positively charged Lys surprisingly provided similar phenotypes as the negatively charged Glu residue. Computational chemistry analysis indicated that the propensity for the C-terminal segment of ECL2b to form an extended a-helix was increased from 15% in the wild type to 89% and 82% by introduction in position 204(C+6) of a Glu or a Lys residue, respectively. Moreover, the constitutive activity of the receptor was inhibited by Zn(2+) binding in an engineered metal-ion site stabilizing an a-helical conformation of this loop segment. It is concluded that the high constitutive activity of the ghrelin receptor is dependent upon flexibility in the C-terminal segment of extracellular loop 2 and that mutations or ligand binding which constrains this segment and thereby conceivably TM-V relative to TM-III movements inhibits the high constitutive signaling.
AB - Based on a rare, natural Glu for Ala204(C+6) variant located six residues after the conserved Cys residue in extracellular loop 2 (ECL2b) associated with selective elimination of the high constitutive signaling of the ghrelin receptor, this loop was subjected to a detailed structure functional analysis. Introduction of Glu in different positions demonstrated that although the constitutive signaling was partly reduced when introduced in position 205(C+7) it was only totally eliminated in position 204(C+6). No charge-charge interaction partner could be identified for Glu(C+6) variant despite mutational analysis of a number of potential partners in the extracellular loops and outer parts of the transmembrane segments. Systematic probing of position 204(C+6) with amino acid residues of different physicochemical properties indicated that a positively charged Lys surprisingly provided similar phenotypes as the negatively charged Glu residue. Computational chemistry analysis indicated that the propensity for the C-terminal segment of ECL2b to form an extended a-helix was increased from 15% in the wild type to 89% and 82% by introduction in position 204(C+6) of a Glu or a Lys residue, respectively. Moreover, the constitutive activity of the receptor was inhibited by Zn(2+) binding in an engineered metal-ion site stabilizing an a-helical conformation of this loop segment. It is concluded that the high constitutive activity of the ghrelin receptor is dependent upon flexibility in the C-terminal segment of extracellular loop 2 and that mutations or ligand binding which constrains this segment and thereby conceivably TM-V relative to TM-III movements inhibits the high constitutive signaling.
U2 - 10.1074/jbc.M112.383240
DO - 10.1074/jbc.M112.383240
M3 - Journal article
C2 - 22846991
VL - 287
SP - 33488
EP - 33502
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
ER -
ID: 38542234