The tyrosine within the NPXnY motif of the human angiotensin II type 1 receptor is involved in mediating signal transduction but is not essential for internalization

SA Laporte, G Servant, DE Richard, E Escher, G Guillemette and R Leduc

Department of Pharmacology, Faculty of Medicine, Universite de Sherbrooke, Quebec, Canada.

The NPXnY motif is involved in the internalization process of several types of receptors, including lipoprotein receptors and G protein- coupled receptors. We replaced Tyr302 with either phenylalanine or alanine in the NPLFY site of the human angiotensin II receptor type 1 and determined the pharmacological properties of the resulting mutant receptors. Competitive binding experiments revealed that COS-7 cells transfected with either the wild-type or mutant receptors expressed approximately the same amount of high affinity binding sites (Bmax 70,000 sites/cell and Kd approximately 2 nM). Photoaffinity labeling of both native and mutant receptors revealed apparent molecular masses of 110 kDa. Incubation of transfected cells with 0.2 nM [125I]Ang II at 37 degrees revealed an efficient internalization of the wild-type receptor and the mutant receptors, although the mutant receptors were internalized at a slower rate. Interestingly, however, the transmembrane signaling was severely impaired in transfected cells expressing mutant receptors. No significant production of inositol- 1,4,5-trisphosphate was observed when these cells were challenged for 3 min with a concentration of angiotensin II as high as 1 microM. This is in contrast to the dose-dependent stimulation of inositol-1,4,5- trisphosphate production in cells expressing the wild-type receptor. Thus, our results show that the Tyr302 in the NPXnY motif of the human angiotensin II receptor type 1 is not essential for agonist binding properties or for internalization of the receptor but plays an important role in transmembrane signaling.

Volume 49, Issue 1, pp. 89-95, 01/01/1996
Copyright © 1996 by American Society for Pharmacology and Experimental Therapeutics

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