Characterization of endothelin receptors in mesangial cells: evidence for two functionally distinct endothelin binding sites

MS Simonson and A Rooney

Department of Medicine, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.

Endothelin (ET) peptides are growth factors that bind to G protein- coupled receptors and serve as a useful model to study mitogenic signal transduction by vasoactive peptides. To begin to define the molecular mechanisms underlying mitogenic signaling by ET-1, we analyzed expression of ET receptor subtypes in glomerular mesangial cells and antagonism of ET-1-induced mitogenic responses by ET receptor antagonists. Competitive displacement analysis of 125I-ET-1 binding revealed a shallow multicomponent curve consistent with the presence of two ET-1 binding sites (Kd values of 32 pM and 1.2 nM). Nonlinear regression analysis demonstrated that a two-site model fit the data better than a one-site model (p = 0.0063). Analysis of 125I-ET-1 binding sites by affinity cross-linking revealed incorporated radioactivity in two distinct protein bands in mesangial cell membranes. Both ETA-specific (BQ 123) and nonselective (PD 142893/PD 145065) receptor antagonists displaced 125I-ET-1 from the low affinity site. The Ki values for BQ 123 and PD 145065 were similar to the IC50 values for inhibition of ET-1-induced increases in the intracellular free Ca2+ concentration ([Ca2+]i) by these antagonists. The ETB- specific ligands S6c and [Ala1,3,11,15]ET-1(6-21) were unable to displace 125I-ET-1 from either low affinity or high affinity binding sites. Analysis of ET receptor mRNA by reverse transcription-polymerase chain reaction, using primers predicted from DNA sequences conserved through evolution in ETA and ETB genes, demonstrated that mesangial cells express a canonical ETA receptor. Collectively, these data suggest that the low affinity, high capacity 125I-ET-1 binding site is an ETA receptor and that the high affinity, low capacity site is not accounted for by canonical ET receptors. We further demonstrated that BQ 123 and PD 142893/PD 145065 inhibited ET-1-stimulated [3H]thymidine uptake but at higher concentrations than required for inhibition of [Ca2+]i increases. Preincubation (as opposed to coincubation) with antagonists was required to inhibit ETA-mediated increases in [Ca2+]i produced by ET-1. These results suggest that the unique, nearly irreversible binding of ET-1 to ETA receptors explains why high concentrations of ET receptor antagonists are required to block longer term actions such as mitogenesis.

Volume 46, Issue 1, pp. 41-50, 07/01/1994
Copyright © 1994 by American Society for Pharmacology and Experimental Therapeutics

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