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Department of Pharmacology, University of California San Diego, La Jolla, California
Received December 12, 2003; accepted December 12, 2003.
PubMed identifies 85,590 citations for the combined keyword set of angiotensin, angiotensinogen, renin, and angiotensin-converting enzyme, yet much remains to be elucidated about the mammalian angiotensin system(s) despite nearly 100 years of research (Bader et al., 2001
). This reflects several facets of this small peptide autocoid. First, angiotensin II has been linked with a diverse set of functions essential for normal mammalian physiology and apparently contributes to a variety of clinical disorders. Second, multiple tissue angiotensin systems exist, each operating through different downstream mechanisms yet often resulting in the same end physiological effects. Third, this area of research has led to multiple billion-dollar pharmaceuticals, and several of the original patents have or will soon expire, leading to efforts to find new proprietary materials. Fourth, it's a fun system to work with, because getting answers to experimental questions is not very difficult but interpreting and extrapolating the results becomes the main challenge. Last, but not least, genomic studies indicate that much of the interindividual variation in expression or functioning of this system, as well as response to system-directed pharmaceuticals, may reside in or be dictated by genetic variants (e.g., polymorphisms) (Baudin, 2002). It is this last facet that holds the promise for the future of angiotensin research and probably for all of pharmacology.
Efforts to link the angiotensin system to human disorders, primarily hypertension, has been ongoing since the discovery of renin and angiotensin. We know of two human angiotensin II receptor subtypes, AT1 and AT2, encoded by different genes, whereas rodents have two variants of the AT1 subtype, AT1A and AT1B (Clauser et al., 1996). The majority of currently known actions of angiotensin are attributed to the AT1 receptor, whereas functions of the AT2, apart from its actions in the central nervous systems and during development, remain somewhat controversial (Rosenstiel et al., 2002; Wagenaar et al., 2002). With the sequencing of angiotensin, pharmacologists and biochemists began to probe structure-function relationships of what is now known as the AT1 subtype by synthesizing thousands of sequence variants of angiotensin II and measuring affinity, efficacy, and peptide conformation. Such studies led to the development of specific antagonists and identification of critical amino acid side chains necessary for potency and efficacy. Indirectly, they also provided the first views of receptor topography. These studies were soon complemented by measurements of AT1 signal transduction pathways and all together established the AT1 (and AT2) as a classic G protein-coupled receptor. Cloning of the receptors opened the door to mutagenesis studies, and soon receptor amino acids were being attributed with receptor functions—ligand binding, internalization, signal transduction, etc. What was somewhat dismaying to investigators interested in function-disorder relationships was the finding that the AT1 receptor coupled to many alternative signaling pathways (Yin et al., 2003), leading some investigators to question why everyone was not hypertensive!
Molecular genetics opened the door for investigators to start analyzing gene sequences in search of polymorphisms, and the angiotensin system was one of the first to be probed. Single nucleotide polymorphisms (SNPs) were identified in angiotensinogen, angiotensin-converting enzyme, and the AT1 receptor genes and population geneticists undertook association studies to test relationships of these SNPs to different cardiovascular disorders, including hypertension (Hopkins and Hunt, 2003). The initial results were chaotic; inconsistency in replication became the initial resounding message (Jeunemaitre and Gimenez-Roqueplo, 2002; Koopmans et al., 2003; Zhu and Cooper, 2003). This reflected in part the contributions of genetic ethnicity and in part the fact that SNPs may be silent or neutral and without overt influence on gene product or gene-function relationships. That mutations or genetic polymorphisms may not translate into altered gene functioning has been appreciated by geneticists for years, yet such conclusions were less familiar to bench molecular biologists who could point to studies in which single nucleotide mutagenesis resulted in significantly altered receptor functioning in vitro and even in vivo. Thus, many published single base changes are mainly of academic interest and should not be extrapolated as potential disease contributors. What has been lacking for the pharmacologist was the frequency of occurrence of SNPs, around 8 ± 2 per 10 kilobases for humans (Zhao et al., 2003a), and knowledge of the gene polymorphisms that existed in nature (in humans or model organisms). The shift of the human and model organism genome projects to construction of databases of SNPs, construction of SNP maps, and recently to construction of haplotype blocks has changed the scene for all pharmacologists. Genomics and molecular and population genetics, which have been circling the "field of engagement" for the past 10 years, have arrived at center stage of molecular pharmacology—and our world will never be the same!
The current issue contains a report from Hansen et al. (2004) detailing studies of a series of AT1 receptor variants engineered to replicate reported coding polymorphisms (cSNPs) identified in a series of different population genetic studies. These cSNPs were examined originally for association with different human (cardiovascular) disorders ranging from genetic hypertension, diabetic nephropathy, renal dysfunction, coronary constriction, and left ventricular hypertrophy to cerebrovascular disease (see article for references). The experimental logic behind the current study was beautifully simple. First, an assumption was made that nonsynonymous polymorphisms (i.e., those that result in coding changes) would more probably be associated with altered gene product functions and diseases. This was based on an earlier study by the same group (Lee et al., 2003). Second, given that these cSNPs of the AT1 receptor have been established within subsets of human populations with disease (albeit at a minor allelic frequency), if one created the equivalent receptor gene variant, expressed it in a defined in vitro cell system and tested it for altered receptor properties—ligand affinity, efficacy, and signal transduction—one could discern the relative importance of each polymorphism to receptor function. Thus, results of epidemiological association studies were converted into classic hypothesis-driven bench research. Of course, this is not the first time that a human SNP was replicated in an in vitro model for study. The difference here is that the study design was to compare and contrast a set of cSNPs all from a single gene.
Seven receptor gene variants were synthesized and tested, first in a high-throughput cellular screening assay (termed receptor selection and amplification technology), then by direct cellular ligand binding, and finally for altered signaling focusing on inositol phosphate and extracellular signal-regulated kinase phosphorylation assays as downstream readouts. Three of the cSNPs were within transmembrane (TM) spanning domains of the receptor, one each in TM1, TM5, and TM6, whereas four were in the intracellular carboxyl terminus (Fig. 1). The receptor selection and amplification technology assay identified the three TM polymorphisms as different from wild type, whereas the four carboxyl terminal polymorphisms seemed neutral. Of the three TM coding changes, none interfered with cell surface expression, whereas one, G45R in TM1, completely abolished receptor affinity and efficacy. A cysteine-to-tryptophan mutation (C289W) reduced potency but not efficacy, whereas a phenylalanine-to-serine mutation (F204S) reduced both potency and efficacy. Thus, all three coding changes could be classified as leading to "loss of function". The glycine-to-arginine polymorphism at position 45 was first reported by Rolfs et al. (1994) from a study of human control subjects, with no evident cardiovascular disease in the test subjects or first-degree relatives. This site is localized in the TM1 domain near the first intracellular loop. Given that the change is from a polar amino acid with minimal volume demands to a charged, extended side chain amino acid, it is perhaps not surprising that it disrupts key receptor functions, such as ligand binding and transduction. Although the authors speculate that the G45R polymorphism may be determined (in the future) to be a "disease-related" (polymorphism) for AT1 receptor physiology, it is unclear why the "control" subjects who carried this polymorphism exhibit no evident pathology. In rodents, the AT1B can subserve many of the functions of the AT1A in mice devoid of AT1A (Oliverio et al., 1997); however, AT1A-null mice exhibit hypertrophy of the juxtaglomerular apparatus and glomerular mesangial expansion along with evidence of a "survival disadvantage" (Oliverio et al., 1998). As far as is known, loss of the AT1 in humans would not be compensated for by an alternate AT1 variant.
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Hansen et al. (2004) raise more questions than answers, as good research should. Would single polymorphisms of membrane receptors be expected to exert incrementally measurable effects on receptor function? Data from the current article could argue in either direction. The four coding changes in the carboxyl terminus did not alter measured receptor properties, implying that a single cSNP may indeed be neutral and not additive: however, what if other receptor functions were altered, but were missed in this study because they were not measured? Thus, what is the most appropriate battery of in vitro tests for subtly altered receptor functions? The C289W polymorphism reduced only potency, which agrees with studies showing that this segment of the receptor polypeptide interacts with peptide agonist (Perodin et al., 2002), whereas the F204S polymorphism reduced both potency and efficacy. Do these reductions translate into meaningful alterations in organ system or whole organism functions? What evidence would be sufficient to warrant creation of engineered model organisms, and should it be one coding change per strain? More importantly for angiotensin researchers is how "loss of function" translates into altered angiotensin system function? It is correct that changes in potency for the agonist could imply altered pharmacogenomic potential, but disorders associated with the angiotensin system (e.g., genetic hypertension) are generally attributed to abnormal "gain of function." As shown in AT1A receptor knockout studies, "loss of function" could be beneficial in some disorders, not detrimental (Cervenka et al., 2002). Thus, could such coding changes offer an "advantage" to organisms, rather than result in disease?
Pharmacological researchers, whether molecular, physiological or genomic, must now design studies that incorporate knowledge learned from genetic studies and analyses of human and model organisms (including but not limited to rat and mouse). For traits and diseases dictated by complex genetics, individual SNPs are likely to have little impact from a population genetic standpoint (Zhao et al., 2003b). Rather, haplotypes, and perhaps even combinations of haplotypes in different genes, are probably needed to answer the ultimate question of what underlies real biological variability (Gabriel et al., 2002). If this is the case, future studies, like the current one, should focus on in vitro haplotype constructs so as to better replicate potential disease processes or complex traits. Finally, Hansen et al. (2004) illustrate that in the broad picture of pharmacological research, the role and importance of genetic background in experimental design must begin to be taken into consideration. Genetic background effects can introduce significant confounds if the question being asked extends to the whole cell, organ system, or beyond (Le Corvoisier et al., 2003; Monti et al., 2003; Printz et al., 2003). These and other lessons gleaned from Hansen et al. (2004) argue the dawn of a new era, and a very challenging one at that.
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Footnotes |
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Address correspondence to: Dr. Morton P. Printz, Department of Pharmacology, 0636, University of California San Diego, 9500 Gilam Drive, Basic Science Bldg., Rm. 3084, La Jolla, CA 92093-0636. E-mail: mprintz{at}ucsd.edu
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