Insertional mutagenesis of the arginine cage domain of the gonadotropin-releasing hormone receptor.
The pattern of side-chain conservation at the cytoplasmic side of the third transmembrane domain of rhodopsin family G protein-coupled receptors, Asp/Glu-Arg-Tyr/X-X-X-Ile/Val, defines a structural "arginine cage" domain. Previous computational and mutagenesis studies of the GnRH receptor indicated an important contribution of local interactions to the function of this domain. We have investigated the functional importance of the intrahelical position and orientation of the arginine cage using insertional mutagenesis. Introduction of a single Ala proximal to the conserved Asp-Arg of this domain caused loss of detectable ligand binding. Inserting a second Ala, however, restored high-affinity agonist binding. Further insertion of three or four Ala residues at this site generated receptors that bound agonist with an affinity 3- to 10-fold higher than that of the wild-type receptor. Loss of detectable coupling to inositol phosphate turnover in all these mutant receptors confirms that the structure required in this region for efficient signaling is highly constrained. In contrast, the recovery of agonist binding with the progressive insertion of two to four Ala residues indicates that specific orientations of this segment can stabilize high-affinity receptor conformations that are uncoupled from signal transduction.
Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine New York, New York 10029, USA.
S
S
Kitanovic
T
T
Yuen
C A
CA
Flanagan
B J
BJ
Ebersole
S C
SC
Sealfon
D000409:Alanine; D020816:Amino Acid Motifs; D000818:Animals; D001120:Arginine; D019556:COS Cells; D000939:Epitopes / Q000235:genetics / Q000378:metabolism; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D016254:Mutagenesis, Insertional; D017434:Protein Structure, Tertiary; D011966:Receptors, LHRH / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000939:Epitopes; D007295:Inositol Phosphates; C029994:LHRH, Ala(6)-N-Et-ProNH2(9)-iodo-; D011966:Receptors, LHRH; D011994:Recombinant Proteins; D007987:Gonadotropin-Releasing Hormone; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D001120:Arginine; D000409:Alanine