A single amino acid substitution in transmembrane helix VI results in overexpression of the human GnRH receptor.
OBJECTIVE
Construction of constitutively active mutants of the GnRH receptor, a member of the G-protein coupled receptor superfamily, would facilitate investigation of the mechanism of receptor activation.


METHODS
Point mutations were introduced in the human GnRH receptor in positions corresponding to those which caused constitutive activity in other G-protein coupled receptors. The effects of these mutations on ligand binding, receptor intracellular signaling and receptor expression were determined.


METHODS
Wild type and mutated receptor cDNAs were expressed in COS-1 cells. Basal and agonist-stimulated inositol phosphate production and ligand binding were determined. In addition, receptor mRNA levels, cell surface receptor stability and rate of internalization were measured.


CONCLUSIONS
Although none of the mutant receptors exhibited constitutive activity, mutation of Phe-2 72 in transmembrane helix VI to Leu increased cell surface receptor numbers, with unchanged affinities for radiolabeled agonist, superagonist and antagonist peptides compared with wild type receptor. The cell surface receptor stability and rate of internalization were similar for wild type and F272L GnRH receptors. Thus a single amino acid mutation in transmembrane helix VI causes an increase in cell surface receptor numbers, which appears to result from an increased rate of receptor protein translation, processing or insertion into membranes.
Myburgh
D B
DB
Department of Chemical Pathology, University of Cape Town Medical School, Observatory, South Africa.
Pawson
A J
AJ
Davidson
J S
JS
Flanagan
C A
CA
Millar
R P
RP
Hapgood
J P
JP
D019943:Amino Acid Substitution; D000818:Animals; D015152:Blotting, Northern; D019556:COS Cells; D002462:Cell Membrane / Q000737:chemistry; D015870:Gene Expression; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D017433:Protein Structure, Secondary; D011869:Radioligand Assay; D011966:Receptors, LHRH / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
D011966:Receptors, LHRH; D010649:Phenylalanine