Expression of recombinant human follicle-stimulating hormone receptor: species-specific ligand binding, signal transduction, and identification of multiple ovarian messenger ribonucleic acid transcripts.
The ligand specificity and biochemical properties of the human (h) FSH receptor are poorly characterized due to the low abundance of these receptors and the limited availability of human tissues. Using a fragment of rat FSH receptor cDNA, we screened a human testicular cDNA library and obtained a FSH receptor cDNA covering the entire amino acid-coding region. After transfection of a human fetal kidney cell line (293) with the hFSH receptor cDNA, radioligand receptor analysis revealed the presence of high affinity (Kd, 1.7 x 10(-9) M) FSH-binding sites on the plasma membrane. Both recombinant and wild-type hFSH displaced [125I]hFSH binding, with ED50 values of 25 and 70 ng/ml, respectively, whereas hLH, hCG, and hTSH were ineffective. Although human, rat(r), and ovine FSH as well as equine CG competed for rat testicular FSH receptor binding, only hFSH and rFSH interacted effectively with the recombinant hFSH receptor, suggesting that species-specific ligand recognition exists between human and rodent receptors. After incubation of transfected cells with hFSH, but not recombinant hLH or hCG, a dose-dependent increase (ED50, 10 ng/ml) in extracellular cAMP accumulation was observed, indicating a functional coupling of the expressed human receptor with the endogenous adenyl cyclase. In cells cotransfected with the FSH receptor expression plasmid and a luciferase reporter gene driven by the promoter of a cAMP-responsive gene, treatment with hFSH, but not hCG, resulted in a dose-dependent increase in luciferase activity. Northern blot analysis using a cRNA probe derived from the human receptor cDNA indicated the presence of multiple FSH receptor mRNA transcripts (7.0, 4.2, and 2.5 kilobases) in RNA prepared from human follicular phase ovary, but not from human corpus luteum or placenta. Additionally, two FSH-binding sites of 76 and 112 kilodaltons were detected in transfected 293 cells after ligand/receptor cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These results demonstrate the expression of functional hFSH receptor with unique ligand specificity and provide new data on the biochemical properties of the human receptor at the mRNA and protein levels.
Tilly
J L
JL
Department of Gynecology and Obstetrics, Stanford University School of Medicine, California 94305-5317.
Aihara
T
T
Nishimori
K
K
Jia
X C
XC
Billig
H
H
Kowalski
K I
KI
Perlas
E A
EA
Hsueh
A J
AJ
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D015152:Blotting, Northern; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D000242:Cyclic AMP / Q000096:biosynthesis; D005260:Female; D005640:Follicle Stimulating Hormone / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression; D006736:Horses; D006801:Humans; D008156:Luciferases / Q000235:genetics; D010053:Ovary / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D011962:Receptors, FSH / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D012756:Sheep; D015398:Signal Transduction; D013045:Species Specificity; D014162:Transfection
D003432:Cross-Linking Reagents; D012333:RNA, Messenger; D011962:Receptors, FSH; D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP; D008156:Luciferases