Bioassay for growth hormone releasing hormone (GHRH) using a recombinant receptor and cAMP-responsive reporter system

Julie A. Frank-Heinrich, Yuannan Xia, Prabhjit Chadha-Mohanty, Fred W. Wagner, H. Edward Grotjan

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

Growth hormone releasing hormone (GHRH) receptors are members of the G- protein receptor family that use cAMP as a second messenger. A human fetal kidney 293-derived cell line stably expressing the porcine GHRH receptor (pGHRHr/293 cells) and a cAMP-responsive reporter system were used to develop a bioassay for human GHRH. The reporter system (phα180SEAP) was constructed by subcloning the tandem cAMP response elements from the human glycoprotein hormone α subunit gene promoter (hα180) upstream from the secreted alkaline phosphatase cDNA of reporter plasmid pSEAP-Basic. To generate a stable cell line expressing both the GHRH receptor and SEAP reporter system, a DNA fragment from pPUR that confers puromycin resistance was subcloned downstream from the reporter construct of phα180SEAP. Tranfection of phα180SEAPpur into pGHRHr/293 cells yielded pGHRHr/SEAP/293 cell lines that responded to recombinant GHRH with dose-dependent increases in SEAP activity. The GHRH receptor-SEAP reporter bioassay was compared to a conventional bioassay using cultured rat anterior pituitary cells. Synthetic and recombinant GHRH induced a 3.1-fold increase in growth hormone release by rat pituitary cells with ED50's of 3.6 and 2.2 x 10-10 M, respectively. Recombinant GHRH was 1.7 ± 0.7 times more potent than synthetic GHRH in the pituitary cell bioassay. In an analogous experiment, pGHRHr/SEAP/293 cells responded to synthetic and recombinant GHRH with a 9.1-fold increase in SEAP activity. The ED50's were 7.8 and 4.3 x 10-11 M, respectively, with recombinant GHRH being 1.8 ± 0.1 times more potent than the synthetic preparation. Thus, the GHRH receptor-SEAP reporter bioassay is a sensitive, accurate, precise and efficient method for measuring GHRH biological activity.

Original languageEnglish (US)
Pages (from-to)65-72
Number of pages8
JournalMolecular and Cellular Endocrinology
Volume150
Issue number1-2
DOIs
StatePublished - Apr 25 1999

Keywords

  • Bioassay
  • CAMP-responsive
  • GHRH
  • Receptor
  • SEAP reporter system

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Endocrinology

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