Characterization of growth hormone-releasing hormone (GHRH) binding to cloned porcine GHRH receptor

• Published in: Peptides (New York, N.Y. : 1980) N.Y. : 1980), 1995, Vol.16 (8), p.1469-1473
• Main Authors: Hassan, Hazem A., Hsiung, Hansen M., Zhang, Xing-Yue, Smith, Dennis P., Smiley, David L., Heiman, Mark L.
• Format: Article
• Language: English
• Subjects: Animals; Binding, Competitive; Cell Line; Cloned receptor; Cloning, Molecular; GHRH binding; GHRH receptors; Growth hormone-releasing hormone (GHRH); Growth Hormone-Releasing Hormone - analogs & derivatives; Growth Hormone-Releasing Hormone - metabolism; Guanine Nucleotides - pharmacology; Humans; In Vitro Techniques; Kinetics; Porcine GHRH receptor; Rats; Receptors, Neuropeptide - drug effects; Receptors, Neuropeptide - genetics; Receptors, Neuropeptide - metabolism; Receptors, Pituitary Hormone-Regulating Hormone - drug effects; Receptors, Pituitary Hormone-Regulating Hormone - genetics; Receptors, Pituitary Hormone-Regulating Hormone - metabolism; Swine; Transfection
• ISSN: 0196-9781; 1873-5169
• DOI: 10.1016/0196-9781(95)02026-8

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 [His 1, 125I-Tyr 10,Nle 27]hGHRH(1–32)-NH 2 increased linearly with protein concentration (10–45 μg protein/tube). Binding reached equilibrium after 90 min at 30°C and remained constant for at least 240 min. Binding was reversible to one class of high-affinity sites ( K d = 1.04 ± 0.19 n M, B max = 3.9 ± 0.53 pmol/mg protein). Binding was selective with a rank order of affinity (IC 50) for porcine GHRH (2.8 ± 0.51 n M), rat GHRH (3.1 ± 0.69 n M), [ N-Ac-Tyr 1, d-Arg 2]hGHRH(3–29)-NH 2 (3.9 ± 0.58 n M), and [ d-Thr 7]GHRH(1–29)-NH 2 (189.7 ± 14.3 n M), 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.