CONTRIBUTIONS OF ARGININE-43 AND ARGININE-94 OF HUMAN CHORIOGONADOTROPIN-BETA TO RECEPTOR-BINDING AND ACTIVATION AS DETERMINED BY OLIGONUCLEOTIDE-BASED MUTAGENESIS

被引:36
作者
CHEN, F [1 ]
PUETT, D [1 ]
机构
[1] UNIV MIAMI,SCH MORPHOL,DEPT BIOCHEM & MOLEC BIOL,REPROD SCI & ENDOCRINOL LABS D5,POB 016960,MIAMI,FL 33101
关键词
D O I
10.1021/bi00106a014
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Members of the glycoprotein hormone family contain a common a subunit and a hormone-specific beta-subunit. Human choriogonadotropin (hCG) beta is a 145 amino acid residue protein glycosylated at 6 positions (2 N-linked and 4 O-linked oligosaccharides). In an effort to elucidate receptor determinants on hCG-beta, we have used site-directed mutagenesis to prepare and express several mutant cDNAs with replacements at arginines-43 and -94. Arg-43 is invariant in all known mammalian CG/lutropin beta-amino acid sequences, and Arg-94 is conserved in 10 of the 12 sequences. Moreover, various studies involving synthetic peptides and enzymatic digestions of intact beta-chains suggest that these residues may be important in hCG receptor binding. Point mutants were made in which these two arginines were replaced with the corresponding residues in human follitropin-beta, Leu-43 and Asp-94. The wild-type and mutant beta-chains were expressed in CHO cells containing a stably integrated gene for bovine-alpha, and heterodimer formation occurred. These heterologous gonadotropins were active in assays using transformed Leydig cells, competitive binding with standard I-125-hCG, and cAMP and progesterone production, but the potency was considerably less than that associated with the hCG-beta wild-type-containing gonadotropin. The double-mutant protein Arg-43 to Leu/Arg-94 to Asp also associated with bovine-alpha, but the resultant heterodimer exhibited only low activity. Replacement of each arginine with lysine yielded heterodimers that were at least as potent as bovine alpha-hCG-beta wild type, but the Lys-43-containing beta-chain appeared to exhibit a low degree of subunit association or reduced stability relative to the expressed hCG-beta wild type. These results demonstrate that arginines-43 and -94 contribute to receptor binding through a positive charge.
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页码:10171 / 10175
页数:5
相关论文
共 26 条
[1]   CHARACTERIZATION OF SEVERAL CLONAL LINES OF CULTURED LEYDIG TUMOR-CELLS - GONADOTROPIN RECEPTORS AND STEROIDOGENIC RESPONSES [J].
ASCOLI, M .
ENDOCRINOLOGY, 1981, 108 (01) :88-95
[2]  
BIRKEN S, 1987, ENDOCRINOLOGY, V112, P657
[3]  
BOUSFIELD GR, 1988, J BIOL CHEM, V263, P12602
[4]   CONVERSION OF HUMAN CHORIOGONADOTROPIN INTO A FOLLITROPIN BY PROTEIN ENGINEERING [J].
CAMPBELL, RK ;
DEANEMIG, DM ;
MOYLE, WR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1991, 88 (03) :760-764
[5]   SPARE GONADOTROPIN RECEPTORS IN RAT TESTIS [J].
CATT, KJ ;
DUFAU, ML .
NATURE-NEW BIOLOGY, 1973, 244 (137) :219-221
[6]   HIGH-FREQUENCY TRANSFECTION OF CHO CELLS USING POLYBRENE [J].
CHANEY, WG ;
HOWARD, DR ;
POLLARD, JW ;
SALLUSTIO, S ;
STANLEY, P .
SOMATIC CELL AND MOLECULAR GENETICS, 1986, 12 (03) :237-244
[7]  
CHEN F, 1991, J BIOL CHEM, V266, P6904
[8]  
CHEN F, 1990, 72ND END SOC M ATL
[9]  
COLOMA TAS, 1990, J BIOL CHEM, V265, P5037
[10]   A SYNTHETIC PEPTIDE CORRESPONDING TO HUMAN-FSH-BETA-SUBUNIT 33-53 BINDS TO FSH RECEPTOR, STIMULATES BASAL ESTRADIOL BIOSYNTHESIS, AND IS A PARTIAL ANTAGONIST OF FSH [J].
COLOMA, TAS ;
DATTATREYAMURTY, B ;
REICHERT, LE .
BIOCHEMISTRY, 1990, 29 (05) :1194-1200