Scanning-alanine mutagenesis of long loop residues 33-53 in follicle stimulating hormone beta subunit.
Follicle stimulating hormone (FSH) is a gonadotropin and member of the pituitary/placental glycoprotein hormone family which bind to G-protein-coupled receptors. These hormones are heterodimers composed of a common alpha and distinct beta -subunits. Previous experimental evidence suggested that the FSH beta -subunit long loop comprised of amino acids Tyr33 to Phe53 is involved in receptor binding and activation and in subunit interaction. According to recently reported crystal structures of human chorionic gonadotropin (hCG), the homologous long loop of the beta -subunit of hCG associates with the alpha -subunit and is partially exposed to solvent. This report describes the results of scanning alanine mutagenesis used to determine if amino acid side chains in this region of the molecule are required for receptor binding and/or subunit contact. Five mutations were made which spanned this loop and the mutant FSH beta-subunits were co-expressed with alpha-subunit in a Baculovirus-infected insect-cell expression system. Mutation of 48QKTCT52 to 48AAACA52 produced a FSH beta-subunit that failed to form heterodimer, consistent with the crystal structure of hCG which shows these amino acids are buried at the subunit interface. The four remaining mutants produced heterodimer and were assayed for binding to and activation of human FSH receptors. Mutation of 37LVY39 to 37AAA39 caused a 20-fold reduction binding (ID50 of 7.0 nM compared with 0.3 nM for wildtype). Mutation of 34TRDL37 to 34AAAA37 or 44RPKI47 to 44APAA47 caused lesser but measurable effects with ID50 values of 1.1 nM and 1.9 nM, respectively. The (40)KDPA(43) to 40KDPA43 to 40AAPA43 mutation had little effect on receptor binding (ID50 = 0.5 nM).
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Wadsworth Center, New York State Department of Health, Albany, 12201-0509, USA.
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D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D005640:Follicle Stimulating Hormone / Q000737:chemistry / Q000235:genetics*; D043373:Follicle Stimulating Hormone, Human; D037201:Follicle Stimulating Hormone, beta Subunit; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics*; D051381:Rats; D011962:Receptors, FSH / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D018076:DNA, Complementary; D043373:Follicle Stimulating Hormone, Human; D037201:Follicle Stimulating Hormone, beta Subunit; D010446:Peptide Fragments; D011962:Receptors, FSH; C072584:follicle stimulating hormone, beta subunit (33-53), human; D005640:Follicle Stimulating Hormone; D000409:Alanine