Human Angiotensin II Type 1 Receptor Isoforms Encoded by Messenger RNA Splice Variants Are Functionally Distinct

Human Angiotensin II Type 1 Receptor Isoforms Encoded by Messenger RNA Splice Variants Are Functionally Distinct Mickey M. Martin, Barry M. Willardson, Gregory F. Burton, C. Roger White, Joseph N. McLaughlin, Steven M. Bray, James W. Ogilvie, Jr., and Terry S. Elton Department of Chemistry and Biochemistry (M.M.M., B.M.W., J.N.M., S.M.B., J.W.O., T.S.E.) Department of Microbiology (G.F.B.) Brigham Young University Provo, Utah 84602 University of Alabama at Birmingham Vascular Biology and Hypertension Program (C.R.W.) Birmingham, Alabama 35294 lar smooth muscle cells (2–4), renal mesangial cells (5), cardiomyocytes (6), and cardiac fibroblasts (7). This mitogenic response requires the rapid activation of one or several mitogen-activated protein kinases including extracellular signal-regulated kinases 1/2 (ERK 1/2), stress-activated C-Jun N-terminal kinases, and p38 mitogen-activated protein kinase (8). The biological responses to Ang II are mediated by its interaction with high affinity G protein-coupled receptors (GPCRs) localized on the surface of target cells (9). Two main Ang II receptor subtypes, AT1R and AT2R, have been pharmacologically identified (10). AT1R activation by Ang II stimulates phosphatidylinositol-specific phospholipase C, leading to the generation of inositol trisphosphate and diacylglycerol, which are involved in intracellular Ca2⫹ mobilization (11, 12) and protein kinase C activation (13). AT1R activation by Ang II also stimulates the ERK 1/2 cascade (14); however, the coupling mechanisms between the AT1R and the ERK 1/2 cascade are still incompletely characterized. Recent investigations suggest that Ang II activates ERK 1/2 through transactivation of tyrosine kinase receptors, which appears to be mediated by several nonreceptor tyrosine kinases, including the proline-rich tyrosine kinase 2 (PYK2) and Src family tyrosine kinases (14–21). Transactivation results in Shc-Grb2-SOS complex formation and RAS activation, which in turn initiates a kinase cascade culminating in ERK 1/2 activation (22, 23). In contrast, the signaling pathways of the AT2R are not well defined. Although the exact physiological function of the AT2R is not clear, studies utilizing vascular smooth muscle cells (24) or coronary endothelial cells (25) suggest that the AT2R inhibits proliferation. Thus, the AT2R may antagonize the growth-promoting effects of the AT1R. Recently, our laboratory (26, 27) and others (28, 29) have demonstrated that the human AT1R (hAT1R) gene is Human tissues that express the angiotensin II (Ang II) type 1 receptor (hAT1R) can synthesize four distinct alternatively spliced hAT1R mRNA transcripts. In this study, we show that the relative abundance of these mRNA transcripts varies widely in human tissues, suggesting that each splice variant is functionally distinct. Here we demonstrate, for the first time, that the hAT1R-B mRNA splice variant encodes a novel long hAT1R isoform in vivo that has significantly diminished affinity for Ang II (i.e. >3fold) when compared with the short hAT1R isoform (encoded by hAT1R-A mRNA splice variant). This reduced agonist affinity caused a significant shift to the right in the dose-response curve for Ang II-induced inositol trisphosphate production and Ca2ⴙ mobilization of the long hAT1R when compared with that of the short hAT1R. The functional differences between these isoforms allows Ang II responsiveness to be fine-tuned by regulating the relative abundance of the long and short hAT1R isoform expressed in a given human tissue. (Molecular Endocrinology 15: 281–293, 2001) INTRODUCTION The peptide hormone, angiotensin II (Ang II), the biologically active component of the renin-angiotensin system, regulates a variety of physiological responses including fluid homeostasis, aldosterone production, renal function, and contraction of vascular smooth muscle (1). Additionally, Ang II has been demonstrated to be a growth-promoting factor in cultured rat vascu0888-8809/01/$3.00/0 Molecular Endocrinology 15(2): 281–293 Copyright © 2001 by The Endocrine Society Printed in U.S.A. 281 MOL ENDO · 2001 282 comprised of at least four exons and spans greater than 60 kilobases (kb). Exons 1, 2, and 3 have been presumed to constitute the 5⬘-untranslated region (UTR) mRNA sequence, while exon 4 harbors the entire uninterrupted open reading frame, for the hAT1R. A comparison of several published hAT1R cDNA sequences revealed that although these cDNA clones shared identical open reading frames, they differed in portions of their presumed 5⬘-UTR (30–32). These results suggested that alternative splicing events combine various 5⬘-UTR exons (i.e. exons 1–3) with the same coding region exon (i.e. exon 4). In support of this hypothesis, our laboratory demonstrated by 5⬘-rapid amplification of cDNA ends (RACE) experiments that four distinct hAT1R mRNA splice variants are synthesized in human lung tissues (i.e. hAT1R mRNA transcripts are comprised of exons 1 and 4; exons 1, 3, and 4; exons 1, 2, and 4; or exons 1, 2, 3, and 4) (26, 27). Sequence analysis has shown that an AUG triplet located in exon 3 is in frame with the downstream open reading frame located in exon 4 (29). Therefore, hAT1R mRNA transcripts containing exons 3 and 4 may encode a novel hAT1R with an amino-terminal extension of 32 amino acids (long hAT1R) when compared with the short receptor encoded by exon 1, 4 hAT1R mRNA. Curnow et al. (29) have previously demonstrated that the exon 1,3,4 hAT1R mRNA transcript was expressed in a number of human tissues. Additionally, they demonstrated that human kidney 293 cells transfected with an exon 1,3,4/hAT1R expression construct produced a functional hAT1R (29). Although these investigators demonstrated that transfected 293 cells express hAT1Rs, they were unable to determine whether these cells were actually expressing the long hAT1R isoform. This is a critical consideration since the AUG codon harbored in exon 3 is not a consensus Kozak translation initiation start site (33). Therefore, it is possible that the hAT1R mRNA exon 1,3,4 splice variant does not encode the long hAT1R, but rather encodes the short hAT1R, since translation may only be initiated at the previously characterized AUG start codon harbored in exon 4 (26–29). Therefore, the following study was initiated to determine whether the long hAT1R is actually expressed in vivo and, if so, to determine whether the long and short hAT1R isoforms are pharmacologically and functionally distinct. RESULTS Tissue Distribution of hAT1R mRNA Splice Variants Four known distinct alternatively spliced hAT1R mRNAs are synthesized from a single hAT1R gene. These transcripts are comprised of exons 1 and 4 (hAT1R-A); exons 1, 3, and 4 (hAT1R-B); exons 1, 2, and 4 (hAT1R-C); and exons 1, 2, 3, and 4 (hAT1R-D) (Fig. 1) (26, 27). These alternatively spliced mRNAs differ only in the lengths of their 5⬘-UTR encoded by exons 1, 2, and 3 while exon 4 harbors the open Vol. 15 No. 2 Fig. 1. A (...truncated)