Interaction of Endothelin-1 with Cloned Bovine ETA Receptors:  Biochemical Parameters and Functional Consequences

• Published in: Biochemistry (Easton) Vol. 35; no. 47; pp. 14868 - 14875
• Main Authors: Desmarets, Julien, Gresser, Olivia, Guedin, Denis, Frelin, Christian
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 26.11.1996
• Subjects: Animals; Cattle; Cell Line; Cloning, Molecular; Endothelin-1 - metabolism; Enzyme Activation; Fibroblasts; Receptor, Endothelin A; Receptors, Endothelin - genetics; Receptors, Endothelin - metabolism; Type C Phospholipases - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi961238w

This paper defines the properties of interaction of endothelin-1 (Et-1) with cloned bovine ETA receptors. The K d value of Et-1/ETA receptor complexes was estimated in membrane preparations to 20 pM using kinetic experiments and saturation experiments performed under quasi equilibrium conditions. Competition experiments yield a wide range of apparent K d(Et-1) values from 20 pM to 1 nM which were in fact measures of the receptor concentrations rather than of K d values. This resulted from the fact that complex second-order rate kinetics rather than pseudo-first-order kinetics control the association of Et-1 to its receptor when the receptor concentration is larger than K d(Et-1). Et-1 induced a production of inositol phosphates with an apparent affinity of 2.3 nM, 100 times higher than the K d(Et-1) value determined previously. Numerical simulation suggested that under time-limited conditions, sub-nanomolar rather than picomolar concentrations of Et-1 are necessary to occupy an important fraction of picomolar sites. It is concluded that bovine ETA receptors have a single affinity state for Et-1 (K d = 20 pM) and that this affinity state can account for nanomolar actions of Et-1 in intact cells. It is suggested that the sensitivity of a preparation to Et-1 is a cell property rather than a receptor property. It is also suggested that the main advantage of high-affinity Et-1 binding is to promote autocrine actions rather than a high potency of the peptide.