Contributions of arginines-43 and -94 of human choriogonadotropin. beta. to receptor binding and activation as determined by oligonucleotide-based mutagenesis

Journal Article · Tue Oct 22 00:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00106a014· OSTI ID:5488529

Chen, Fang; Puett, D ^[1]

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1. Univ. of Miami, FL (United States)

Members of the glycoprotein hormone family contain a common {alpha} 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}, the authors 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 {sup 125}I-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 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.

• OSTI ID: 5488529
• Journal Information: Biochemistry; (United States), Vol. 30:42; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
GONADOTROPINS
RADIORECEPTOR ASSAY
RECEPTORS
BIOCHEMICAL REACTION KINETICS
ARGININE
GLYCOPROTEINS
IODINE 125
LEUCINE
MUTAGENESIS
OLIGONUCLEOTIDES
RECOMBINANT DNA
AMINO ACIDS
BETA DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
DAYS LIVING RADIOISOTOPES
DNA
ELECTRON CAPTURE RADIOISOTOPES
HORMONES
INTERMEDIATE MASS NUCLEI
INTERNAL CONVERSION RADIOISOTOPES
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
MEMBRANE PROTEINS
NUCLEI
NUCLEIC ACIDS
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PEPTIDE HORMONES
PITUITARY HORMONES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
TRACER TECHNIQUES
550201* - Biochemistry- Tracer Techniques