Development of a Homologous Radioimmunoassay for Mouse Growth Hormone Receptor*

Abstract

Abstract A RIA for mouse GH receptor (mGHR) was developed. A synthetic peptide corresponding to the carboxyl-terminal 14 amino acids of the mGHR (GHR-2 peptide) was used as the antigen for antiserum production. The synthetic 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 from virgin and 15-day-pregnant mice. Serial dilutions of these samples yielded displacement curves parallel to the synthetic peptide. No significant cross-reactivity was seen with serum from virgin or 15-day-pregnant mice, mGH, recombinant mGH-binding protein (mGHBP), a synthetic peptide 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 significantly 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 during 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 total number of binding sites in livers from virgin and pregnant mice using the GH RRA and the combined results of the mGHR and mGHBP RIAs showed that the two methods gave almost identical results for livers from virgin 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 mGHR plus mGHBP RIAs, respectively. However, in livers from pregnant animals, the combined results from the mGHR and mGHBP RIAs were approximately 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/mg protein (n = 3) for the mGHR plus the mGHBP RIAs and the GH RRA, respectively. The increase in the total GH binding capacity in livers from pregnant mice compared with those from virgin animals was largely due 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) in 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 virgin animals to 6.369 ± 0.607 pmol/mg protein (n = 3) in pregnant mice.