Bioassay for growth hormone releasing hormone (GHRH) using a recombinant receptor and cAMP-responsive reporter system.
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 (ph alpha180SEAP) was constructed by subcloning the tandem cAMP response elements from the human glycoprotein hormone alpha subunit gene promoter (h alpha180) 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 ph alpha180SEAP. Tranfection of ph alpha180SEAPpur 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.
Animal Science Department, University of Nebraska, Lincoln 68583-0908, USA.
J A
JA
Frank-Heinrich
Y
Y
Xia
P
P
Chadha-Mohanty
F W
FW
Wagner
H E
HE
Grotjan
D000818:Animals; D001681:Biological Assay; D002460:Cell Line; D000242:Cyclic AMP; D013007:Growth Hormone-Releasing Hormone / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051381:Rats; D018013:Receptors, Neuropeptide / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D018017:Receptors, Pituitary Hormone-Regulating Hormone / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D018013:Receptors, Neuropeptide; D018017:Receptors, Pituitary Hormone-Regulating Hormone; D011994:Recombinant Proteins; D013007:Growth Hormone-Releasing Hormone; D000242:Cyclic AMP; C048250:somatotropin releasing hormone receptor