title: Site-directed mutagenesis of amino acids 33-44 of the common alpha-subunit reveals different structural requirements for heterodimer expression among the glycoprotein hormones and suggests that cyclic adenosine 3',5'-monophosphate production and growth promotion are potentially dissociable functions of human thyrotropin.
abstract: Amino acid residues 33-44 of the common alpha-subunit of the glycoprotein hormones have been implicated in heterodimerization as well as high affinity receptor binding of human (h) CG. In the present study, we compared the role of specific amino acids within this region for glycoprotein hormone heterodimer formation, using a transient transfection system to coexpress different mutant alpha-subunit constructs with the beta-subunit of either hTSH, hCG, or hFSH. Our results identified a crucial role for alpha Pro38 in the heterodimer expression of hTSH as well as hFSH, similiar to what had been described for hCG. In contrast, alpha Ala38, which had been critical for hCG, was not essential for hTSH heterodimer expression and less important for hFSH, whereas alpha Phe33 and alpha Arg35 appeared uniquely important for hFSH. Furthermore, we assessed the role of these residues for bioactivity and receptor binding of hTSH. Mutation of the surface-exposed residues alpha Arg42-Ser43-Lys44, which form part of a unique alpha-helical structure, to Ala42-Ala43-Ala44, decreased TSH receptor binding using porcine thyroid membranes as well as rat FRTL-5 cells. Residues alpha Phe33 and alpha Arg35, in contrast, were not important for high affinity binding of hTSH. In the signal transduction of hTSH, alpha Ala36 was necessary for efficient growth induction in FRTL-5 cells but not for cAMP production in either FRTL-5 cells or Chinese hamster ovary cells expressing the human TSH receptor (JP09). Similarly, residues alpha Arg42-Ser43-Lys44 were more important for hTSH-mediated induction of cell growth than cAMP production. Mutating alpha Arg35 to Ala reduced cAMP induction but not receptor binding of hTSH. In summary, using site-directed mutagenesis, we identified a domain, residues 33-44 of the common alpha-subunit, important in heterodimer expression, receptor binding, and activation of hTSH. The comparison of the relative roles of specific amino acids within this region in hTSH with hCG and hFSH highlights previously unrecognized differences in the structural requirements for heterodimer expression among the members of the glycoprotein hormone family. Moreover, our findings revealed a novel role for residues alpha 33-44 in triggering different postreceptor events, suggesting that cAMP production and growth promotion may, at least in part, be dissociable functions of hTSH.
authors.affiliation: Molecular and Cellular Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health Bethesda, Maryland 20892-1758, USA.
authors.forename: M
authors.initials: M
authors.lastname: Grossmann
authors.forename: M W
authors.initials: MW
authors.lastname: Szkudlinski
authors.forename: J A
authors.initials: JA
authors.lastname: Dias
authors.forename: H
authors.initials: H
authors.lastname: Xia
authors.forename: R
authors.initials: R
authors.lastname: Wong
authors.forename: D
authors.initials: D
authors.lastname: Puett
authors.forename: B D
authors.initials: BD
authors.lastname: Weintraub
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D002455:Cell Division / Q000235:genetics; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D051381:Rats; D011989:Receptors, Thyrotropin / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013961:Thyroid Gland / Q000166:cytology / Q000378:metabolism; D013972:Thyrotropin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D006063:Chorionic Gonadotropin; D011989:Receptors, Thyrotropin; D011994:Recombinant Proteins; D013972:Thyrotropin; D000242:Cyclic AMP