DEVELOPMENT OF A HOMOLOGOUS RADIOIMMUNOASSAY FOR MOUSE GROWTH-HORMONERECEPTOR

A RIA for mouse GH receptor (mGHR) was developed. A synthetic peptide corresponding to the carboxyl-terminal 14 amino acids of the mGHR (GHR -3 peptide) was used as the antigen for antiserum production. The synt hetic peptide was also used as the standard and radioligand in the RIA . The ability of the antiserum to recognize the mGHR was demonstrated by quantitating receptor concentrations in liver and mammary gland fro m virgin and 15-day-pregnant mice. Serial dilutions of these samples y ielded displacement curves parallel to the synthetic peptide. No signi ficant cross-reactivity was seen with serum from virgin or 15-day-preg nant mice, mGH, recombinant mGH-binding protein (mGHBP), a synthetic p eptide identical to the hydrophilic tail of mGHBP, or a 14-amino acid synthetic peptide corresponding to amino acids 338-351. of mGHR (GHR-1 peptide). The concentration range of the mGHR RIA was 0.5-200 nM, and the intra- and interassay coefficients of variation were 6.5% and 6.1 %, respectively. The concentration of liver GHR increased significantl y during pregnancy compared with that in virgin mice, from 0.246 +/- 0 .045 pmol/mg protein (mean +/-: SEM; n = 5) in the virgin animals to 1 .015 +/- 0.159 pmol/mg protein (n = 5) in pregnant mice. In contrast, the mGHR concentration in the mammary gland decreased significantly du ring pregnancy from 0.606 +/- 0.201 pmol/mg protein (mean +/- SEM; n = 5) to 0.299 +/- 0.027 pmol/mg protein (n = 5). Comparison of the tota l number of binding sites in livers from virgin and pregnant mice usin g the GH RRA and the combined results of the mGHR and mGHBP RIAs showe d that the two methods gave almost identical results for livers from v irgin animals, or 0.363 +/- 0.063 pmol/mg protein (mean +/- SEM; n = 3 ) and 0.371 +/- 0.008 pmol/mg protein (n = 3) for the GH RRA and the m GHR plus mGHBP RIAs, respectively. However, in livers from pregnant an imals, the combined results from the mGHR and mGHBP RIAs were approxim ately 1.8 times higher than those obtained by the GH RRA, or 6.732 +/- 0.612 pmol/mg protein (mean +/- SEM; n = 3) and 3.693 +/- 0.67 pmol/m g protein (n = 3) for the mGHR plus the mGHBP RIAs and the GH RRA, res pectively. The increase in the total GH binding capacity in livers fro m pregnant mice compared with those from virgin animals was largely du e to an increase in the GHBP content. The increase in GHR was only 2.4 -fold, or from 0.153 +/- 0.01 pmol/mg protein (mean +/-: SEM; n = 3) i n virgin mice to 0.364 +/- 0.03 pmol/mg protein (n = 3) in the 15-day- pregnant mice, whereas GHBP increased almost 30-fold during pregnancy, or from 0.218 +/- 0.003 pmol/mg protein (mean +/- SEM; n = 3) in virg in animals to 6.369 +/- 0.607 pmol/mg protein (n = 3) in pregnant mice .