Characterization of growth hormone-releasing hormone (GHRH) binding to cloned porcine GHRH receptor.
To study structure-activity relationships of growth hormone-releasing hormone (GHRH), a competitive binding assay was developed using cloned porcine adenopituitary GHRH receptors expressed in human kidney 293 cells. Specific binding of [His1, 125I-Tyr10,Nle27]hGHRH(1-32)-NH2 increased linearly with protein concentration (10-45 micrograms protein/ tube). Binding reached equilibrium after 90 min at 30 degrees C and remained constant for at least 240 min. Binding was reversible to one class of high-affinity sites (Kd = 1.04 +/- 0.19 nM, Bmax = 3.9 +/- 0.53 pmol/mg protein). Binding was selective with a rank order of affinity (IC50) for porcine GHRH (2.8 +/- 0.51 nM), rat GHRH (3.1 +/- 0.69 nM), [N-Ac-Tyr1, D-Arg2]hGHRH(3-29)-NH2 (3.9 +/- 0.58 nM), and [D-Thr7]GHRH(1-29)-NH2 (189.7 +/- 14.3 nM), consistent with their binding to a GHRH receptor. Nonhydrolyzable guanine nucleotides inhibited binding. These data describe a selective and reliable method for a competitive GHRH binding assay that for the first time utilizes rapid filtration to terminate the binding assay.
Hassan
H A
HA
Division of Endocrinology, Eli Lilly & Company, Indianapolis, IN 46285, USA.
Hsiung
H M
HM
Zhang
X Y
XY
Smith
D P
DP
Smiley
D L
DL
Heiman
M L
ML
D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D003001:Cloning, Molecular; D013007:Growth Hormone-Releasing Hormone / Q000031:analogs & derivatives / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D051381:Rats; D018013:Receptors, Neuropeptide / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018017:Receptors, Pituitary Hormone-Regulating Hormone / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D014162:Transfection
D006150:Guanine Nucleotides; D018013:Receptors, Neuropeptide; D018017:Receptors, Pituitary Hormone-Regulating Hormone; D013007:Growth Hormone-Releasing Hormone; C048250:somatotropin releasing hormone receptor