Scanning-alanine mutagenesis of long loop residues 33–53 in follicle stimulating hormone beta subunit

Abstract

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 α and distinct β-subunits. Previous experimental evidence suggested that the FSHβ-subunit long loop comprised of amino acids Tyr 33 to Phe 53 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 β-subunit of hCG associates with the α-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 FSHβ-subunits were co-expressed with α-subunit in a Baculovirus-infected insect-cell expression system. Mutation of 48QKTCT 52 to 48AAACA 52 produced a FSHβ-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 37LVY 39 to 37AAA 39 caused a 20-fold reduction in receptor binding (ID 50 of 7.0 nM compared with 0.3 nM for wildtype). Mutation of 34TRDL 37 to 34AAAA 37 or 44RPKI 47 to 44APAA 47 caused lesser but measurable effects with ID 50 values of 1.1 nM and 1.9 nM, respectively. The 40KDPA 43 to 40AAPA 43 mutation had little effect on receptor binding (ID 50 = 0.5 nM). Each of the mutant heterodimers could activate human FSH receptors as measured by progesterone production in an in vitro bioassay. These results corroborate the notion that the hFSHβ long loop contains amino acids involved in subunit association as shown in the hCG model. This loop also contains residues not previously identified ( 37LVY 39) whose side chains affect receptor interaction and steroidogenesis.