Elsevier

Peptides

Volume 16, Issue 8, 1995, Pages 1469-1473
Peptides

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

https://doi.org/10.1016/0196-9781(95)02026-8Get rights and content

Abstract

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 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 (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.

References (22)

  • A.R. Friedman et al.

    Degradation of growth hormone releasing factor analogs in neutral aqueous solution is related to deamidation of asparagine residues

    Int. J. Pept. Protein Res.

    (1991)
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