Endocrinology, Vol 136, 4377-4381, Copyright © 1995 by Endocrine Society

ARTICLES

Binding and cross-linking of 32P-labeled human relaxin to human uterine cells and primary rat atrial cardiomyocytes

PL Osheroff and KL King
Department of Protein Chemistry, Genentech, Inc., South San Francisco, California 94080, USA.

Relaxin is known for its function in parturition and has been suggested to participate in the regulation of blood pressure, heart rate, and the release of neuropeptides such as oxytocin and vasopressin. Consistent with the physiological roles of relaxin, high affinity relaxin receptors have been demonstrated in the rat uterus, brain, and cardiac atrium. Here we report the binding and cross-linking of a biologically active, 32P-labeled human relaxin to a human uterine cell line and primary rat atrial cardiomyocytes. Relaxin binding to the human uterine cells consisted of a single class of high affinity sites (Kd = approximately 0.44 nM) with approximately 1082 +/- 62 binding sites/cell. Binding and cross-linking of relaxin to the human uterine cells and rat atrial cardiomyocytes followed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis analysis revealed that the putative relaxin receptor showed a major component with an apparent M(r) greater than 220 kilodaltons and a minor component of approximately 36 kilodaltons, and was not disulfide linked. The binding and cross- linking of [32P]relaxin could be displaced by unlabeled relaxin in a concentration-dependent manner, but not by a 1000-fold molar excess of insulin, insulin-like growth factor I (IGF-I), or IGF-II. These data suggested that the relaxin receptor was similar in size but distinct from the insulin, IGF-I, and IGF-II receptors.

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