Recognition of follicle stimulating hormone (alpha-subunit) by a recombinant receptor protein domain coded by an alternately spliced mRNA and expressed in Escherichia coli.
To assess the functional significance of putative proteins encoded by alternately spliced mRNA of the sheep testicular FSH receptor, a short form cDNA comprising of the first four exons (117 residues mature protein) was engineered for expression in Escherichia coli. The expressed protein of molecular mass 15 kDa was purified to homogeneity and verified by reaction with an antibody against a synthetic peptide sequence unique to the amino (N)-terminal region FSH receptor. The purified FSH receptor domain protein bound 125I-labeled hFSH in a ligand blot on polyvinylidine difluoride membranes. Further analyses by slot blot revealed high affinity of the immobilized protein with significant reaction at 10 pmol. As the immobilized receptor protein also reacted with structurally related hormones (125I-labeled LH/125I-labeled human chorionic gonadotropin), we confirmed that interaction most probably occurred via the common alpha-subunit of these glycoprotein hormones. Our results reveal that this N-terminal portion of the FSH receptor contain(s) major site(s) for hormone recognition that could be mediated via the alpha-subunit. A rabbit antibody to the receptor inhibited FSH action in receptor bearing cells, revealing the utility of such recombinant FSH receptor protein(s) for modulation of hormone action.
Khan
H
H
Molecular Reproduction Research Laboratory, Clinical Research Institute of Montreal, Quebec, Canada.
Jiang
L G
LG
Jayashree
G N
GN
Yarney
T A
TA
Sairam
M R
MR
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D015292:Glycoprotein Hormones, alpha Subunit / Q000378:metabolism*; D015151:Immunoblotting; D008024:Ligands; D008297:Male; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism*; D011962:Receptors, FSH / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D012756:Sheep
D000906:Antibodies; D018076:DNA, Complementary; D015292:Glycoprotein Hormones, alpha Subunit; D008024:Ligands; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D011962:Receptors, FSH; D011994:Recombinant Proteins