Aspartic acid in the second transmembrane domain is a highly conserved amino acid among the G proteincoupled receptors and is functionally important for agonist binding and G-protein coupling in P2-adrenergic and luteinizing hormone receptors. To determine whether this aspartic acid is also involved in the function of the rat vascular angiotensin II (AII) have been distinguished on the basis of their differential affinity for two classes of antagonists, DUP753 and CGP42112A or PD123177, respectively (1, 2). The AT1 receptor is responsible for the majority ofAll actions, including vasoconstriction, aldosterone secretion, and growthpromoting effects (1, 4, 5). The structure and potential function of the AT2 receptor are still unknown.The proteins encoded by the cDNAs for rat (6), bovine (7), human (8, 9), and mouse (10) AT1 receptors contain seven a-helical transmembrane domains and belong to the G protein-coupled receptor superfamily. Two closely related isoforms (ATia and ATlb) encoded by different genes have been identified in rat and mouse species (10-12), whereas in other species only one AT1 receptor has been identified so far. The intracellular signaling pathway of AII through the AT1 receptors includes coupling to a GTP-binding protein, activation of a phospholipase C resulting in inositol trisphosphate generation, and mobilization of intracellular Ca2+ stores and diacylglycerol formation leading to protein kinase C activation (13).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Previous data strongly suggest that the ligand-binding domain involves mainly polar residues of the seven transmembrane helices (14,15). An aspartate residue located in the second transmembrane domain is conserved in all AT1 receptors and in all of the receptors with peptide ligands cloned to date (vasopressin, bradykinin, somatoliberin, tachykinin peptides, etc.), with the exception of the substance P receptor (glutamate instead of aspartate). Its replacement by an asparagine in the j32-adrenergic receptor (Asp79 Asn) (16)

The capacity of the angiotensin II (AngII) agonist [Sarl]AngII, the antagonist [Sari-Ile8]AngII and the non-peptidic antagonist DuP753 to undergo receptor internalization were studied in Chinese hamster ovary cells expressing rat AngII type la or 1 b receptors (AT,, or ATlb) or a mutant of AT,, (Asn") unable to couple G-protein. In this expression system, the ligand-induced internalization of rat AT,, and AT,, are similar. Moreover, peptidic ligands, either the agonist or antagonist, induce a significant internalization of AT, receptors, but the non-peptidic antagonist DuP753 is far less potent. Finally, the normal internalization of the mutant Asn74 demonstrates that receptor activation and G-protein coupling are not required for AT,, internalization.

Two distinct types of cell-surface angiotensin II receptors (AT1 and AT2) have been defined pharmacologically and cDNAs encoding each type have been identified by expression cloning. These pharmacological studies showed the AT1 receptors to mediate all the known functions of angiotensin II in regulating salt and fluid homeostasis. Further complexity in the angiotensin II receptor system was revealed when homology cloning showed the existence of two AT1 subtypes in rodents and in situ hybridization and reverse transcription-polymerase chain reaction analyses showed their level of expression to be regulated differently in different tissues: AT1A is the principal receptor in the vessels, brain, kidney, lung, liver, adrenal gland and fetal pituitary, while AT1B predominates in the adult pituitary and is only expressed in specific regions of the adrenal gland (zona glomerulosa) and kidney (glomeruli). Expression of AT1A appears to be induced by angiotensin II in vascular smooth-muscle cells but is inhibited in the adrenal gland. Preliminary analysis of the AT1 promoters is also suggestive of a high degree of complexity in their regulation. Investigation of a potential role for altered AT1 receptor function has commenced at a genetic level in several diseases of the cardiovascular system. No mutations affecting the coding sequence have been identified in Conn adenoma and no linkage has been demonstrated with human hypertension by sib-pair analysis. None the less, certain polymorphisms that do not alter the protein structure have been found to be associated with hypertension and to occur at an increased frequency in conjunction with specific polymorphisms in the ACE gene in individuals at increased risk for myocardial infarction. Further characterization of the regions of the AT1 gene that regulate its expression are therefore needed. The physiological importance of the AT2 gene product still remains a matter of debate.