The functional genomics of guanylyl cyclase natriuretic peptide receptor-a: Perspectives and paradigms

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1 MINIREVIEW The functional genomics of guanylyl cyclase natriuretic peptide receptor-a: Perspectives and paradigms Kailash N. Pandey Department of Physiology, Tulane University Health Sciences Center School of Medicine, New Orleans, LA, USA Keywords cardiac hypertrophy; functional genomics; guanylyl cyclase receptor; hypertension; natriuretic peptides Correspondence K. N. Pandey, Department of Physiology, SL 39, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA Fax: Tel: (Received 2 September 2010, revised 7 December 2010, accepted 2 March 2011) doi: /j x The cardiac hormones atrial natriuretic peptide and B-type natriuretic peptide (brain natriuretic peptide) activate guanylyl cyclase (GC)-A natriuretic peptide receptor-a (NPRA) and produce the second messenger cgmp. GC-A NPRA is a member of the growing family of GC receptors. The recent biochemical, molecular and genomic studies on GC-A NPRA have provided important insights into the regulation and functional activity of this receptor protein, with a particular emphasis on cardiac and renal protective roles in hypertension and cardiovascular disease states. The progress in this field of research has significantly strengthened and advanced our knowledge about the critical roles of Npr1 (coding for GC-A NPRA) in the control of fluid volume, blood pressure, cardiac remodeling, and other physiological functions and pathological states. Overall, this review attempts to provide insights and to delineate the current concepts in the field of functional genomics and signaling of GC-A NPRA in hypertension and cardiovascular disease states at the molecular level. Introduction The initial work by de Bold et al. [1] established that atrial extracts contained natriuretic and diuretic activities, and demonstrated the existence of atrial natriuretic factor atrial natriuretic peptide (ANP). Members of a family of endogenous peptide hormones including atrial natriuretic factor ANP, B-type natriuretic peptide (brain natriuretic peptide) (BNP), C-type natriuretic peptide (CNP) and urodilatin are considered to play an integral role in hypertension and cardiovascular regulation via their ability to mediate excretion of sodium and water, reduce blood volume, and elicit a vasorelaxation effect [2 5]. Interestingly, the natriuretic peptide hormones have been suggested not only to regulate blood pressure but also to play a role in a number of additional processes, namely: antimitogenic effects, inhibition of myocardial hypertrophy, endothelial cell function, cartilage growth, immunity, and mitochondrial biogenesis [6 9]. ANP and BNP are also increasingly being utilized to screen and diagnose cardiac etiologies for shortness of breath and congestive heart failure (CHF) in emergency situations [10]. One of the principal loci involved in the regulatory action of ANP and BNP is that encoding the receptor guanylyl cyclase (GC)-A, designated GC-A natriuretic peptide receptor-a (NPRA). Interaction of ANP and BNP with GC-A NPRA produces the intracellular Abbreviations BNP, B-type natriuretic peptide; CHF, congestive heart failure; CNP, C-type natriuretic peptide; GC, guanylyl cyclase; GCD, guanylyl cyclase catalytic domain; IP 3, inositol trisphosphate; KHD, protein kinase-like homology domain; LVH, left ventricular hypertrophy; MAPK, mitogenactivated protein kinase; NPRA, natriuretic peptide receptor-a; NPRB, natriuretic peptide receptor-b; NPRC, natriuretic peptide receptor-c; PDE, cgmp-dependent phosphodiesterase; PKG, cgmp-dependent protein kinase; RAA, renin angiotensin aldosterone; VSMC, vascular smooth muscle cell FEBS Journal 278 (2011) ª 2011 The Author Journal compilation ª 2011 FEBS

2 K. N. Pandey Update on functional aspects of GC-A NPRA second messenger cgmp, which plays a central role in the pathophysiology of hypertension and cardiovascular disorders [5,11,12]. Gaining insights into the intricacies of ANP NPRA signaling is of pivotal importance for understanding both receptor biology and the disease state arising from abnormal hormone receptor interactions. It has been postulated that the binding of ANP to the extracellular domain of the receptor causes a conformational change, thereby transmitting the signal to the GC catalytic domain (GCD); however, the exact mechanism of receptor activation remains unknown. Recent studies have focused on elucidating, at the molecular level, the nature and mode of functioning of GC-A NPRA. Both cultured cells in vitro and genetargeted mouse models in vivo have been utilized to gain a better understanding of the normal and abnormal control of cellular and physiological processes. Although there has been much appreciation of the functional roles of natriuretic peptides and their cognate receptors in renal, cardiovascular, endocrine and skeletal homeostasis; in-depth research studies are still needed to fully understand their potential molecular targets in cardiovascular and other disease states. Ultimately, it is expected that studies on the natriuretic peptides and their receptors should yield new therapeutic targets and novel loci for the control and treatment of hypertension and cardiovascular disorders. Natriuretic peptide hormone family ANP is the first described member of the natriuretic peptide hormone family. It is primarily synthesized in the heart atria, and elicits natriuretic, diuretic and vasorelaxant effects, largely directed to the reduction of fluid volume and blood pressure [2,3,5,7,13,14]. Subsequently, BNP and CNP, with biochemical and functional characteristics similar to those of ANP but derived from separate genes, were identified [15]. BNP was initially isolated from the brain; however, it is primarily synthesized in the heart, circulates in the plasma, and displays the most variability in primary structure. CNP is mainly present in endothelial cells, and is highly conserved across species. All three types of natriuretic peptide contain a highly conserved 17-residue disulfide ring, which is essential for the hormonal activities, but they show differences from each other in the N-terminal and C-terminal flanking sequences (Fig. 1). Although ANP has been considered to exert its predominant effects in lowering blood pressure and blood volume, recent evidence indicates that ANP plays a critical role in preventing cardiac load and overgrowth of the heart in pathological conditions. Fig. 1. Comparison of amino acid sequences of the natriuretic peptide hormone family. Comparison of amino acid sequences of human ANP, BNP and CNP with conserved amino acids, which are represented by red boxes. The lines between two cysteines in ANP, BNP and CNP indicate a 17-residue disulfide bridge, which seems to be essential for the biological activity of these peptide hormones. Both ANP and BNP are predominantly synthesized in the heart; ANP levels vary from 50-fold to 100-fold higher than those of BNP. After processing of the 151- residue preprohormone to the 126-residue prohormone, the secretion of proanp is believed to occur predominantly in response to atrial distension [14]. Upon secretion, the cleavage of proanp to generate the active and mature 28-residue ANP molecule is catalyzed by a serine protease, corin [16]. The synthesis and release of ANP from the heart is enhanced in response to various agents and settings, such as arginine vasopressin, endothelin, and vagal stimuli [14,17]. BNP is synthesized as a 134-residue preprohormone, which yields a 108-residue prohormone. Processing of the probnp yields a 75-residue N-terminal BNP and a 32-residue biologically active circulating BNP [18,19]. The atria are the primary sites of synthesis for both hormones within the heart. Although the ventricles also produce both ANP and BNP, the concentrations are 100-fold to 1000-fold less than those in the atria. The expression of both ANP and BNP increases dramatically in both the atria and ventricles in cardiac hypertrophy [20,21]. It is believed that, in the ventricles, BNP synthesis is regulated by volume overload, which activates ventricular wall stretch, subsequently enhancing hormone synthesis at the transcriptional level [22,23]. Interestingly, higher levels of ventricular ANP are present in the developing embryo and fetus, with both mrna and peptide levels of ANP declining rapidly during the prenatal period [24]. CNP is mainly present in the central nervous system [25], vascular endothelial cells [26], and chondrocytes [27]. CNP is synthesized as a 103-residue prohormone, cleaved to a 53-residue peptide by the protease furin, FEBS Journal 278 (2011) ª 2011 The Author Journal compilation ª 2011 FEBS 1793

3 Update on functional aspects of GC-A NPRA K. N. Pandey and subsequently processed to yield the biologically active 22-residue molecule [28]. In addition, a 32-residue peptide termed urodilatin, which is identical to the C-terminal sequence of proanp, is known to be present in urine [29,30]. Urodilatin is not detected in the circulation, and appears to be a unique intrarenal natriuretic peptide with unexplored physiological functions [31]. D-type natriuretic peptide is an additional member of the natriuretic peptide hormone family [32]. DNP is present in the venom of the green mamba (Dendroaspis angusticeps) as a 38-residue peptide. GC Natriuretic peptide receptor family Natriuretic peptides (ANP, BNP, and CNP) bind and activate specific cognate receptors present on the plasma membranes of a wide variety of target cells. Membrane-bound forms of natriuretic peptide receptors have been cloned and sequenced from rat brain [33,34], human placenta [35], and mouse testis [36]. Molecular cloning and expression of cdnas have identified three different forms of natriuretic peptide receptor, including NPRA, natriuretic peptide receptor-b (NPRB), and natriuretic peptide receptor- C (NPRC). These constitute the natriuretic peptide receptor family; however, they show variability in terms of their ligand specificity and signal transduction activity. Two of these receptors contain intrinsic GC activity, and have been designated GC-A NPRA and GC-B NPRB; they are also referred to as GC-A and GC-B, respectively [37 39]. NPRC lacks the GCD, and has been termed a natriuretic peptide clearance receptor; it contains a short (37-residue) cytoplasmic tail, apparently not coupled to GC activation [40]. Both ANP and BNP selectively stimulate NPRA, whereas CNP primarily activates NPRB, and all three natriuretic peptides indiscriminately bind to NPRC [26,39,41]. NPRA is a 135-kDa transmembrane protein, and ligand binding to the receptor generates the second messenger cgmp. It has been suggested that ANP binding to its receptor in vivo requires chloride, which could exert a chloride-dependent feedback-control effect on receptor function [42]. The general topological structure of NPRA is consistent with that seen in the GC receptor family, containing at least four distinct regions: an extracellular ligand-binding domain, a single transmembrane-spanning region, an intracellular protein kinase-like homology domain (KHD), and a GCD [36,37]. NPRB has an overall domain structure similar to that of NPRA, with binding selectivity for CNP [43]. GC-A NPRA is the dominant form of natriuretic peptide receptor found in peripheral organs, and mediates most of the known actions of ANP and BNP. By the use of a homology-based cdna library screening system, additional members of the GC receptor family have also been identified; however, their specific ligand(s) and or activator(s) are not yet known (Table 1). The other members of the GC receptor family are GC-C [11], GC-D [44], GC-E [45], GC-F [45], GC-G [46], retinal GC [47], and GC-Y-X1 [48]. Table 1. Ligand specificity, tissue distribution and gene-disrupted phenotypes of particulate GCs natriuretic peptide receptors. ROS-GC, rod outer segment GC. Receptor Ligand Tissue distribution Gene knockout phenotype in mice GC-A NPRA (Npr1) GC-B NPRB (Npr2) GC-C ANP BNP (Nppa Nppb) CNP (Nppc) Guanylyn, uroguanylyn, enterotoxin Adrenal glands, brain, heart, liver, lung, olfactory glands, ovary, pituitary gland, placenta, testis, thymus, vascular beds, and other tissues Adrenal glands, brain, cartilage, fibroblast, heart, lung, ovary, pituitary gland, placenta, testis, thymus, vascular beds, and other tissues Colon, intestine, kidney GC-D Orphan Neuroepithelium, olfactory glands Unknown [44] GC-E Orphan Pineal gland, retina Unknown [45] GC-F Orphan Retina Unknown [45] GC-G Orphan Intestine, kidney, lung, skeletal muscle, Unknown [46] and other tissues ROS-GC Orphan Rod outer segment Unknown [47] Retinal GC Orphan Retina Unknown [47] GC-Y-X1 Orphan Sensory neurons Unknown [48] High blood pressure, hypertension, cardiac hypertrophy and fibrosis, inflammation, volume overload, reduced testosterone levels [21, ,108,125,126] Dwarfism, decreased adiposity, female sterility, seizures, vascular complication [142,143] Resistance to intestinal secretion, diarrhea [11] 1794 FEBS Journal 278 (2011) ª 2011 The Author Journal compilation ª 2011 FEBS

4 K. N. Pandey Update on functional aspects of GC-A NPRA The intracellular region of NPRA is divided into two domains: the KHD is the 280-residue region immediately following the transmembrane domain, and distal to this is the GCD, which is at the C-terminal portion of the receptor molecule. More than 80% of the conserved residues that have been found in all protein kinases [49] are considered to be present in NPRA [5,6]. The GCD of NPRA has been suggested to consist of a 250-residue region at the C-terminal end of the molecule. Deletion of the C-terminal region of NPRA results in a protein that binds to ANP but does not contain GC activity [38,50,51]. Modeling studies based on the crystal structure of the adenylyl cyclase II C 2 homodimer [52,53] predicted that the active sites of GCs and adenylyl cyclases are closely related [54,55]. On the basis of these predictions, the GC catalytic active site of murine NPRA includes a 31-residue sequence (residues ) at the C-terminal end of the receptor molecule. A comprehensive assessment of the structure function relationship of GC-A NPRA has been described in this series [56]. The transmembrane GC-A NPRA contains a single cyclase catalytic active site per polypeptide molecule; however, modeling data suggest that two polypeptide chains are required to activate the functional receptor [57]. Thus the transmembrane GC receptors seem to function as homodimers [58,59]. The dimerization region of GC-A NPRA has been suggested to be located between the KHD and the GCD, and is predicted to form an amphipathic a-helical structure [58]. NPRB is localized mainly in the brain and vascular tissues, although it is thought to mediate the actions of CNP in the vascular beds and in the central nervous system [43]. The third member of the natriuretic peptide receptor family, NPRC, consists of a large extracellular domain of 496 residues, a single transmembrane domain, and a very short 37-residue cytoplasmic tail that has no homology with any other known receptor protein domain. The extracellular region of NPRC is 30% identical to those of GC-A NPRA and GC-B NPRB. Earlier, it was proposed by default that NPRC functions as a clearance receptor to clear natriuretic peptides from the circulation; however, several studies have also provided evidence that NPRC plays roles in the biological actions of natriuretic peptides [60 62]. Intracellular signal transduction mechanisms of GC-A NPRA ANP markedly increases cgmp levels in target tissues in a dose-related manner [63,64]. The production of cgmp is believed to result from ANP binding to the extracellular domain of NPRA, which probably allosterically regulates an increased specific activity of the cytoplasmic GCD of the receptor molecule [7,51,65,66]. Because the nonhydrolyzable analogs of ATP mimic the effect of ANP, it has been suggested that ATP can allosterically regulate the GC catalytic activity of NPRA [67 70]. In studies with mutant NPRA specifically lacking the KHD, it was found that the mutant receptor was active independently of ANP, which showed that it had the capacity to be bound with ligand, and most importantly, that it had basal GC activity 100-fold greater than that of wild-type NPRA [70]. Those previous findings suggested that, under natural conditions, the KHD acts as a negative regulator of the catalytic moiety of NPRA. Initially, this model was the standard way of explaining the signal transduction mechanism of GC-coupled natriuretic peptide receptors [71]. However, the model has not been supported by the studies of other investigators, which found that deletion of the KHD in NPRA did not cause an elevation of basal GC activity; nevertheless, ATP seems to be obligatory for the transduction activities of both NPRA and NPRB [65,67,72]. It has been suggested that NPRA exists in the phosphorylated form in the basal state, and the binding of ANP causes a decrease in phosphate content as well as a reduction of the ANP-dependent GC activity [73]. This apparent mechanism of desensitization of NPRA is in contrast to what is seen with many other cell surface receptors, which appear to be desensitized by phosphorylation [74 76]. Some previously reported observations have also suggested that the GC activity may, in fact, be regulated by receptor phosphorylation [77 80]. However, little is known about the molecular regulatory mechanisms of the desensitization and signaling pathways of GC-A NPRA, which may involve more than one process. Internalization and sequestration of hormone receptors have been suggested to play important roles in the process of receptor desensitization and downregulation [81]. It is possible that NPRA may undergo homologous desensitization in response to ANP activation that could be mediated by receptor internalization, sequestration, and metabolic degradation, in addition to phosphorylation dephosphorylation mechanisms [82,83]. At the mrna level, NPRA has been shown to be regulated by glucocorticoids [84], transforming growth factor-b [85], chorionic gonadotropin [86], and angiotensin II [87,88]. Endogenous transcription factors such as Ets-1 and p300 have been shown to exert remarkable stimulating effects on Npr1 transcription and expression [89,90]. At the protein level, angiotensin II has been shown to inhibit the GC activity of NPRA [87,91,92]. Similarly, at the receptor level, NPRA is FEBS Journal 278 (2011) ª 2011 The Author Journal compilation ª 2011 FEBS 1795

5 Update on functional aspects of GC-A NPRA K. N. Pandey downregulated following exposure to its ligand ANP or 8-bromo-cGMP [51,64,82,93,94]. Ligand-mediated endocytosis of GC-A NPRA After binding to ANP and BNP, GC-A NPRA is internalized and sequestered into intracellular compartments. Therefore, GC-A NPRA is a dynamic cellular macromolecule that traverses different subcellular compartments during its lifetime. Evidence indicates that, after internalization, the ligand receptor complexes dissociate inside the cell and a population of GC-A NPRA recycles back to the plasma membrane. Subsequently, the dissociated ligands are degraded in the lysosomes. However a small percentage of the ligand escapes the lysosomal degradative pathway and is released intact into the culture medium. GC-A NPRA is internalized into subcellular compartments in a ligand-dependent manner [95 100]. The ligand-dependent endocytosis and sequestration of NPRA involves a series of sequential sorting steps, through which ligand receptor complexes can eventually be degraded. A proportion of receptor is recycled back to the plasma membrane, and a small percentage of intact ligand is released to the cell exterior [51,97,99,100]. The recycling of endocytosed receptor to the plasma membrane and the release of intact ligand to the cell exterior occur simultaneously with processes leading to degradation of the majority of ligand receptor complexes into lysosomes [51,82]. These findings provided direct evidence that treatment of cells with unlabeled ANP accelerates the disappearance of surface receptors, indicating that ANP-dependent downregulation of GC-A NPRA involves internalization of the receptor [82]. All three natriuretic peptides (ANP, BNP, and CNP) are also bind to internalized involving NPRC and ligand-receptor complexes are internalized. The metabolic degradation of natriuretic peptides is further regulated by neprilyisn, as well as by insulin-degrading enzymes, as discussed in this series [101]. The short GDAY motif in the C-terminal domain of GC-A NPRA serves as a signal for endocytosis and trafficking [51,82]. Gly920 and Tyr923 are the critical elements in the GDAY motif. It is thought that Asp921 provides an acidic environment for efficient signaling of the GDAY motif in the internalization of GC-A NPRA. The mutation of Asp921 to alanine did not have a major effect on internalization, but significantly attenuated the recycling of internalized receptors to the plasma membrane [82,83]. On the other hand, mutation of Gly920 and Tyr923 to alanines reduced the internalization of receptor, but did not have any discernible effect on receptor recycling. It was suggested that Tyr923 in the GDAY motif modulates the early internalization of GC-A NPRA, whereas Asp921 seems to mediate recycling or later sorting of the receptor. Increasing evidence indicates that complex arrays of short signals and recognition peptide sequences ensure accurate trafficking and distribution of transmembrane receptors and or proteins and their ligands into intracellular compartments [83,94]. The short signals usually consist of small, linear amino acid sequences, which are recognized by adaptor coat proteins along the endocytic and sorting pathways. In recent years, much has been learned about the function and mechanisms of endocytic pathways responsible for the trafficking and molecular sorting of membrane receptors and their ligands into intracellular compartments; however, the significance and scope of action of the short motifs in these cellular events of GC-A NPRA and GC-NPRB are not well understood. Interestingly, GC-B NPRB is also internalized and recycled in hippocampal neurons and C6 glioma cell cultures [102]. It was suggested that trafficking of GC- B NPRB occurs ligand-dependently in response to CNP binding and stimulation of the receptor protein. The internalization and trafficking of GC-B NPRB has been suggested to involve a clathrin-dependent mechanism. Our recent work indicates that the internalization of GC-A NPRA also involves clathrin-dependent pathways [103]. Receptor internalization is severely diminished by inhibitors of clathrin proteins, such as chlorpromazine and monodensyl cadaverine. However, interaction of the GDAY motif in GC-A NPRA and GC-B NPRB with clathrin adaptor proteins remains to be established. Physiological and pathophysiological functions of GC-A NPRA The interaction of ANP with GC-A NPRA reduces blood volume and lowers blood pressure by enhancing salt and water release through the kidney and inducing vasorelaxation of smooth muscle cells. Both ANP and BNP are implicated in reducing the preload and afterload of the heart in both physiological and pathological conditions. ANP and BNP acting via GC-A NPRA antagonize cardiac hypertrophic and fibrotic growth, thus conferring cardioprotective effects in disease states. ANP has been shown to exert an antimitogenic effect in response to various growth-promoting agonist hormones in a number of target cells and tissues. The binding of ANP and BNP to GC-A NPRA produces increased levels of the intracellular second messenger cgmp, which stimulates three known cgmp effector molecules, namely: cgmp-dependent protein kinases 1796 FEBS Journal 278 (2011) ª 2011 The Author Journal compilation ª 2011 FEBS

6 K. N. Pandey Update on functional aspects of GC-A NPRA (PKGs), cgmp-dependent phosphodiesterases (PDEs), and cgmp-dependent ion channels. The activation of these effector molecules elicits a number of physiological and pathophysiological roles of GC-A NPRA in several target cells and tissue systems (Fig. 2). Thus, multiple synergistic actions of ANP and BNP and their cognate receptor GC-A NPRA make them novel therapeutic targets in renal, cardiac and vascular diseases. The critical physiological and pathophysiological functions of GC-A NPRA are described below. Protective role of GC-A NPRA in blood pressure regulation Genetic mouse models with disruption of both Nppa (coding for proanp) and Npr1 (coding for GC-A NPRA) have provided strong support for the central role of the natriuretic peptide hormone receptor system in the regulation of arterial pressure [21, ]. Therefore, genetic defects that reduce the activity of ANP and its receptor system can be considered as candidate contributors to essential hypertension [7]. Previous studies with ANP-deficient (Nppa ) ) ) mice demonstrated that a defect in proanp synthesis can cause hypertension [107]. The blood pressure of homozygous null mutant mice was elevated by 8 23 mmhg when they were fed with standard-salt or intermediatesalt diets. Those previous findings indicated that genetic disruption of ANP production can lead to hypertension. Transgenic mice overexpressing ANP developed sustained hypotension with an arterial pressure that was mmhg lower than that of their nontransgenic siblings [110,111]. Interestingly, somatic delivery of the ANP gene in spontaneously hypertensive rats induced a sustained reduction of systemic blood pressure [112]. Overexpression of ANP in hypertensive mice lowered systolic blood pressure, raising the possibility of using ANP gene therapy for the treatment of human hypertension [113]. It has also been shown that functional alterations of the Nppa promoter are linked to cardiac hypertrophy in progenies of crosses between Wistar Kyoto and Wistar Kyoto-derived hypertensive rats, and that a singlenucleotide polymorphism can alter the transcriptional activity of the proanp gene promoter [114]. Genetic studies with Npr1 knockout (Npr1 ) ) or zero-copy) mice have indicated that disruption of Npr1 increases blood pressure by mmhg as compared with wild-type (Npr1 + + or two-copy) animals [21,104,109]. It has been demonstrated that complete absence of NPRA causes hypertension in mice and leads to altered renin and angiotensin II levels [21,104,109, ]. In contrast, increased expression Ligand Receptor NPRA NPRB NPRC LBD GTP PKG PDE CNG Phosphoryla on y or dephosphoryla on Ca 2+ IP 3 ANP BNP CNP TM KHD camp DD GCD IP 3 PKC MAPK camp TNF-α IL-6 cgmp GTP cgmp Physiological func ons Sodium excre on Vasodilata on An prolifera on An hypertrophy Fig. 2. Representation of hormone specificity, ligand-binding domains, transmembrane-spanning regions, intracellular domains and signaling systems of GC-A NPRA, GC-B NPRB, and NPRC. The arrows indicate the ligand specificity for specific natriuretic peptide receptors. The extracellular ligand-binding domain (LBD), transmembrane region (TM), KHD and GCD of GC-A NPRA and GC-B NPRB are shown. DD is the dimerization domain of NPRA and NPRB. The LBD, TM and small intracellular tail of NPRC are also indicated. Both NPRA and NPRB have been shown to generate cgmp from the hydrolysis of GTP. An increased level of intracellular cgmp stimulates and activates three known cgmp effector molecules, namely: PKGs, PDEs, and cgmp-dependent ion-gated channels (CNGs). The cgmp-dependent signaling may antagonize a number of pathways, including: intracellular Ca 2+ release, IP 3 formation, activation of protein kinase C (PKC) and MAPKs, and production of cytokines such as tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6). The resulting cascade can mimic ANP NPRA cgmp-dependent responses in both physiological and pathophysiological environments. The activation of NPRC may lead to a decrease in camp levels and an increase in IP 3 production. of NPRA in gene-duplicated mutant mice significantly reduces blood pressure and increases the levels of cgmp, in correspondence with the increasing number of Npr1 copies [106,115,116,118]. Our studies have examined the quantitative contributions and possible mechanisms mediating the responses of varying numbers of Npr1 copies by determining the renal plasma flow, glomerular filtration rate, urine flow and sodium excretion patterns following blood volume expansion in Npr1-targeted mice in a gene dose-dependent manner [105,116]. Our findings demonstrated that the ANP NPRA axis is primarily responsible for mediating the renal hemodynamic and sodium excretory responses to intravascular blood volume expansion. Interestingly, the ANP NPRA system inhibits aldosterone synthesis and release from adrenal glomerulosa FEBS Journal 278 (2011) ª 2011 The Author Journal compilation ª 2011 FEBS 1797

7 Update on functional aspects of GC-A NPRA K. N. Pandey cells [3,109,115,119], which may account for its renal natriuretic and diuretic effects. Furthermore, studies with Npr1-disrupted (zero-copy) mice demonstrated that, at birth, the absence of NPRA allows higher renin and angiotensin II levels than in wild-type mice, and increased renin mrna expression [109]. However, at 3 16 weeks of age, the circulating renin and angiotensin II levels were dramatically decreased in Npr1 homozygous null mutant mice as compared with wild-type (two-copy) control mice. The decrease in renin activity in adult Npr1 null mutant mice is probably caused by a progressive elevation in arterial pressure, leading to inhibition of renin synthesis and release from the kidney juxtaglomerular cells [116]. On the other hand, the adrenal renin content and renin mrna level, as well as angiotensin II and aldosterone concentrations, were elevated in adult homozygous null mutant mice as compared with wild-type mice [109,115]. In light of these previous findings, it can be suggested that the ANP NPRA signaling system may play a key regulatory role in the maintenance of both systemic and tissue levels of the components of the renin angiotensin aldosterone (RAA) system in physiological and pathological conditions. Indeed, ANP NPRA signaling appears to oppose almost all actions of angiotensin II in both physiological and disease states (Table 2). Although expression of ANP and BNP is markedly increased in patients with hypertrophic or failing hearts, it is unclear how the natriuretic peptide system is activated to play a protective role. The ANP NPRA system may act by reducing high blood pressure and inhibiting the RAA system, or by activating new molecular targets as a consequence of the hypertrophic changes occurring in the heart [21,105,120,121]. Table 2. Typical examples of antagonistic actions of ANP NPRA on various angiotensin II-stimulated physiological and biochemical effects in target cells and tissues. CNS, central nervous system; PKC, protein kinase C. Parameters Angiotensin II ANP NPRA Aldosterone release Stimulation Inhibition Renin secretion Inhibition Inhibition Vasopressin release Stimulation Inhibition Blood vessels Contraction Relaxation Water intake Stimulation Inhibition CNS-mediated hypertension Stimulation Inhibition Gonadotropin release Unknown Stimulation Testosterone synthesis Unknown Stimulation Estrodiol synthesis Unknown Stimulation Intracellular Ca 2+ release Stimulation Inhibition MAPKs Stimulation Inhibition PKC Stimulation Inhibition IP 3 production Stimulation Inhibition Functional role of GC-A NPRA and salt sensitivity The disruption of Npr1 indicated that the blood pressure of homozygous mutant mice remained elevated and unchanged in response to either minimal-salt or high-salt diets [122]. These investigators suggested that NPRA may exert its major effect at the level of the vasculature, and probably does so independently of salt. In contrast, other studies reported that disruption of Npr1 resulted in chronic elevation of blood pressure in mice fed with high-salt diets [115,118]. The findings that adrenal angiotensin II and aldosterone levels are increased in Npr1-disrupted mice may explain the elevated systemic blood pressure with decreasing Npr1 copy (zero-copy and one-copy) numbers [115]. However, adrenal angiotensin II and aldosterone levels are decreased in Npr1 gene-duplicated mice. A low-salt diet increased adrenal angiotensin II and aldosterone levels in all Npr1-targeted (gene-disrupted and geneduplicated) mice, whereas a high-salt diet reduced adrenal angiotensin II and aldosterone levels in Npr1- disrupted mice and wild-type mice, but not in Npr1- duplicated (three-copy and four-copy) mice. Our findings suggest that NPRA signaling has a protective effect against high salt in Npr1-duplicated mice as compared with Npr1-disrupted (four-copy) mice [115]. Indeed, more studies are needed to clarify the relationship between salt sensitivity and blood pressures in Npr1-targeted mice. Protective roles of GC-A NPRA in cardiac dysfunction It is believed that ANP and BNP concentrations are markedly increased both in cardiac tissues and in the plasma of CHF patients [ ]. Interestingly, in hypertrophied hearts, ANP and BNP genes are overexpressed, suggesting that autocrine and or paracrine effects of natriuretic peptides predominate, and might serve as an endogenous protective mechanism against maladaptive pathological cardiac hypertrophy [21,120,124, ]. Evidence suggests that a high plasma ANP BNP level is a prognostic predictor in humans with heart failure [123,129]. In patients with severe CHF, concentrations of both ANP and BNP are higher than control values; however, the increase in BNP concentration is 10-fold to 50-fold higher than the increase in ANP concentration [20]. Interestingly, the half-life of BNP is greater than that of ANP; thus, the diagnostic evaluations of natriuretic peptides have favored BNP [125]. The plasma levels of both ANP and BNP are markedly elevated under the pathophysiological conditions of cardiac dysfunction, including 1798 FEBS Journal 278 (2011) ª 2011 The Author Journal compilation ª 2011 FEBS

8 K. N. Pandey Update on functional aspects of GC-A NPRA diastolic dysfunction, CHF, pulmonary embolism, and cardiac hypertrophy [21,124,125,130,131]. It has been suggested that ventricular expression of ANP and BNP is more closely associated with local cardiac hypertrophy and fibrosis than with plasma ANP levels and systemic blood pressure [21,127]. BNP can be considered as an important prognostic indicator in CHF patients; however, N-terminal probnp is considered to be a stronger risk bio-indicator for cardiovascular events [132,133]. The expression of Nppa and Nppb (coding for pro BNP) is greatly stimulated in hypertrophied hearts, suggesting that autocrine and or paracrine effects of natriuretic peptides predominate and might serve as an endogenous protective mechanism against maladaptive cardiac hypertrophy [21,120,134]. Disruption of Npr1 in mice increases the cardiac mass and incidence of cardiac hypertrophy to a great extent [21,104,127, ]. Previous studies have demonstrated that Npr1 disruption in mice provokes enhanced expression of hypertrophic marker genes, proinflammatory cytokines, and matrix metalloproteinases, and enhanced activation of nuclear factor kappab, which seem to be associated with cardiac hypertrophy, fibrosis, and extracellular matrix remodeling [21,126,127]. Interestingly, the expression of sarcolemmal endoplasmic reticulum Ca 2+ -ATPase-2a progressively decreased in the hypertrophied hearts of Npr1 homozygous null mutant mice as compared with wild-type control mice [21]. It has also been demonstrated that expression of angiotensin-converting enzyme and angiotensin II receptor type A is greatly enhanced in Npr1 null mutant (zero-copy) mice as compared with wild-type (two-copy) control mice [127]. Moreover, it has also been suggested that Npr1 antagonizes angiotensin II receptor-mediated and angiotensin II receptor type A-mediated cardiac remodeling, and provides an endogenous protective mechanism in the failing heart [127,138,139]. The arteries of smooth muscle-specific and endothelial cell-specific Npr1 knockout mice exhibited significant arterial hypertension [140]. It has also been suggested that Npr1 represents a potential locus for susceptibility to atherosclerosis [141]. The impact of Npr1 in cardiovascular pathophysiology has also been described in this series [142]. On the other hand, Npr2-deleted mice exhibit dysfunctional endochondral ossification and diminished longitudinal growth in limbs and vertebra, and show normal blood pressure, as compared with their wild-type counterparts [143]. Mutation of Npr2 has been shown to be associated with Maroteaux-type acromesomedic dysplasia [144]. Biological actions of GC-A NPRA in renal and vascular cells ANP NPRA signaling in the kidneys promotes the excretion of salt and water, and enhances glomerular filtration rate and renal plasma flow [3,4,7,116]. Targets of ANP action in the kidney include the inner medullary collecting duct, glomerulus, and mesangial cells [51, ]. The increased production of cgmp at ANP concentrations affecting renal function correlates with the effects of dibutyryl-cgmp, which prevents mesangial cell contraction in response to angiotensin II [148]. ANP markedly lowers renin secretion and also plasma renin concentrations [109,149,150]. The role of ANP in mediating the renal and vascular effects was investigated with selective NPRA antagonists to eliminate the effect of ANP [151,152]. ANP NPRA signaling exerts direct effect on the kidney, to release sodium and water, by inhibiting sodium reabsorption. Npr1 knockout mice exhibit an impaired ability to initiate a natriuretic response to acute blood volume expansion [105]. In Npr1-duplicated mice, a low dose of ANP decreased the fractional reabsorption of distal sodium, suggesting that the augmented natriuresis was enhanced by ANP infusions and is mediated by Npr1 dosage [153]. These findings suggested that ANP NPRA signaling inhibits distal sodium reabsorption. ANP NPRA signaling also exerts indirect effects on renal sodium and water excretion by inhibiting the RAA system, as previously described [5,7]. ANP, either in intact aortic segments or in cultured vascular smooth muscle cells (VSMCs), has always been shown to increase cgmp levels. The correlative evidence of ANP-induced cgmp accumulation has suggested its role as the second messenger of dilatory responses to ANP in cultured VSMCs [152,154,155]. ANP and cgmp analogs reduced the agonist-dependent increases in cytosolic Ca 2+ levels in VSMCs and inositol trisphosphate (IP 3 ) levels in Leydig cells; thus, intracellular cgmp has been suggested to mediate the ANP-induced decrease in cytosolic Ca 2+ and IP 3 levels [156,157]. ANP has also been found to act as a growth suppressor in a variety of cell types, including vasculature, kidney and heart cells, and neurons [51,82,154,155,158]. ANP inhibits mitogen activation of fibroblasts [159], and induces cardiac myocyte apoptosis [160]. However, the mechanisms involved in these effects of ANP are not yet completely understood. Clearly, more studies are warranted to elucidate the molecular mechanisms underlying the antiproliferative effect of ANP NPRA signaling in various target cells. FEBS Journal 278 (2011) ª 2011 The Author Journal compilation ª 2011 FEBS 1799

9 Update on functional aspects of GC-A NPRA K. N. Pandey ANP is considered to be a direct smooth muscle relaxant, and a potent regulator of cell growth and proliferation. It is expected that the antigrowth paradigm could potentially operate through the negative regulation of mitogen-activated protein kinase (MAPK) activities. ANP may be one of the key endogenous hormones that interacts negatively with elements in the MAPK signaling pathway to control cell growth and proliferation. ANP has been reported to antagonize the growth-promoting effects in target cells; however, the mechanism of the antigrowth paradigm of ANP and the involvement of specific ANP receptor subtypes (NPRA and NPRC) in different target cells are controversial [51,62, ]. Association of gene polymorphisms of Nppa, Nppb and Npr1 in hypertension and cardiovascular diseases Recent genetic and clinical studies have indicated an association of Nppa, Nppb and Npr1 polymorphisms with hypertension and cardiovascular events in humans [128, ]. An association between an Nppa promoter polymorphism ( C66UG) and left ventricular hypertrophy (LVH) has been demonstrated in Italian hypertensive patients, indicating that individuals carrying a copy of the Nppa variant allele exhibit a marked decrease in proanp levels associated with LVH [165]. Interestingly, an association between a microsatellite marker in the Npr1 promoter and LVH has also been demonstrated, suggesting that the ANP NPRA system contributes to ventricular remodeling in human essential hypertension [165]. As the relationship between high blood pressure and cardiovascular risk is continuous, in the absence of ANP NPRA signaling even small increases in blood pressure have excessive and detrimental effects. Epidemiological studies have demonstrated that substantial heritability of blood pressure and cardiovascular risks can occur, suggesting a role for genetic factors [167]. Intriguingly, a common genetic variant at the Nppa Nppb locus was found to be associated with circulating ANP and BNP concentrations, contributing to interindividual variations in blood pressure and hypertension [164]. These authors demonstrated that a single-nucleotide polymorphism at the Nppa Nppb locus was associated with increased plasma ANP and BNP concentrations, and lower systolic and diastolic blood pressures. Rare genetic mutations have been suggested for monogenic forms of hypertension and blood pressure in humans [168,169]. However, common variants associated with blood pressure regulation were not established. A number of pathways, namely the RAA system and the adrenergic system, are considered to regulate blood pressure and hypertension; nevertheless, the genetic determinants in these pathways contributing to interindividual differences in blood pressure regulation have not been elucidated. Therefore, the findings of those previous studies indicating an association of common variants in the Nppa Nppb locus with circulating ANP and BNP concentrations are novel [164]. Interestingly, a four-minus haplotype in the 3 -UTR of Npr1 has been shown to be associated with an increased level of N-terminal-proBNP in humans [166]. The four-minus haplotype constitutes 4C repeats at nucleotide position and a 4-bp deletion of AGAA at nucleotide position of Npr1. Individuals with genetic defects in Npr1 caused by the presence of the four-minus haplotype exhibit significantly higher N-terminal probnp levels. It has been speculated that the causal mechanism for this effect could be Npr1 mrna instability, leading to decreased translational production of receptor molecules [170]. This could elicit a feedback mechanism, whereby the diminished function of the BNP NPRA system caused by the defect in Npr1 provokes compensatory enhanced expression and release of BNP. Taken together, these considerations suggest that a positive association exists between Nppa, Nppb and Npr1 polymorphisms and essential hypertension, high blood pressure and left ventricular mass index in humans. Further studies are needed for the characterization of more functionally significant markers of Nppa, Nppb and Npr1 variants in a larger human population. Conclusion and future perspectives The studies outlined in this review provide a unique perspective for delineating the genetic and molecular basis of GC-A NPRA regulation and function. Recent studies have utilized molecular approaches to delineate the physiological functions affected by decreasing or increasing the number of Npr1 copies as achieved by gene targeting, such as gene disruption (gene knockout) or gene duplication (gene dosage), of Npr1 in mice. The gene-targeting strategies have produced mice that contain zero to four copies of the Npr1 locus. Using gene-targeted mouse models, we have been able to determine the effects of decreasing or increasing the expression levels of Npr1 in intact mice in vivo. Comparative analyses of the biochemical and physiological phenotypes of Npr1-disrupted and Npr1-duplicated mutant mice will have enormous potential for answering fundamental questions concerning the biological importance of ANP NPRA signaling in disease states 1800 FEBS Journal 278 (2011) ª 2011 The Author Journal compilation ª 2011 FEBS

10 K. N. Pandey Update on functional aspects of GC-A NPRA by genetically altering Npr1 copy numbers and product levels in vivo in intact animals with otherwise identical genetic backgrounds. The results of these studies have provided important tools for examination of the role of the ANP NPRA system in hypertension and cardiovascular disease states. Future studies will lead to a better understanding of the genetic basis of Npr1 function in regulating blood volume and pressure homeostasis, and should reveal new possibilities for preventing cardiovascular sequelae such as hypertension, heart attack, and stroke. Nevertheless, the paradigms of the molecular basis of the functional regulation of Npr1 and the mechanisms of ANP NPRA action are not yet clearly understood. Currently, natriuretic peptides are considered to be markers of CHF; however, an understanding of their therapeutic potential for the treatment of cardiovascular diseases such as hypertension, renal insufficiency, cardiac hypertrophy, CHF and stroke is still lacking. The results of future investigation should be of great value in resolving the problems of genetic complexities related to hypertension and heart failure. Overall, future studies should be directed at providing a unique perspective for delineating the genetic and molecular basis of Npr1 expression, regulation and function in both normal and disease states. The resulting knowledge should yield new therapeutic targets for treating hypertension and preventing hypertensionrelated cardiovascular diseases and other pathological conditions. Acknowledgements My special thanks go to B. B. Aggarwal, Department of Experimental Therapeutics and Cytokine Research Laboratory, MD Anderson Cancer Center; and to S. L. Hamilton, Department of Molecular Physiology and Biophysics, Baylor College of Medicine, for providing their facilities during our displacement period caused by Hurricane Katrina. I thank my wife Kamala Pandey for her kind help in the preparation of this manuscript. The research work in the author s laboratory was supported by National Institutes of Health grants (HL and HL-62147). References 1 de Bold AJ, Borenstein HB, Veress AT & Sonnenberg H (1981) A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sci 28, de Bold AJ (1985) Atrial natriuretic factor: a hormone produced by the heart. Science 230, Brenner BM, Ballermann BJ, Gunning ME & Zeidel ML (1990) Diverse biological actions of atrial natriuretic peptide. Physiol Rev 70, Levin ER, Gardner DG & Samson WK (1998) Natriuretic peptides. N Engl J Med 339, Pandey KN (2005) Biology of natriuretic peptides and their receptors. Peptides 26, Garbers DL, Chrisman TD, Wiegn P, Katafuchi T, Albanesi JP, Bielinski V, Barylko B, Redfield MM & Burnett JC Jr (2006) Membrane guanylyl cyclase receptors: an update. Trends Endocrinol Metab 17, Pandey KN (2008) Emerging roles of antriuretic peptides and their receptors in pathophysiology of hypertension and cardiovascular regulation. J Am Soc Hypertens 2, Gardner DG (2003) Natriuretic peptides: markers or modulators of cardiac hypertrophy? Trends Endocrinol Metab 14, Richards AM (2007) Natriuretic peptides: update on peptide release, bioactivity, and clinical use. Hypertension 50, Vasan RS, Benjamin EJ, Larson MG, Leip EP, Wang TJ, Wilson PW & Levy D (2002) Plasma natriuretic peptides for community screening for left ventricular hypertrophy and systolic dysfunction: the Framingham heart study. JAMA 288, Lucas KA, Pitari GM, Kazerounian S, Ruiz-Stewart I, Park J, Schulz S, Chepenik KP & Waldman SA (2000) Guanylyl cyclases and signaling by cyclic GMP. Pharmacol Rev 52, Tremblay J, Desjardins R, Hum D, Gutkowska J & Hamet P (2002) Biochemistry and physiology of the natriuretic peptide receptor guanylyl cyclases. Mol Cell Biochem 230, Drewett JG & Garbers DL (1994) The family of guanylyl cyclase receptors and their ligands. Endocr Rev 15, McGrath MF & de Bold AJ (2005) Determinants of natriuretic peptide gene expression. Peptides 26, Rosenzweig A & Seidman CE (1991) Atrial natriuretic factor and related peptide hormones. Annu Rev Biochem 60, Yan W, Wu F, Morser J & Wu Q (2000) Corin, a transmembrane cardiac serine protease, acts as a pro-atrial natriuretic peptide-converting enzyme. Proc Natl Acad Sci USA 97, Thibault G, Amiri F & Garcia R (1999) Regulation of natriuretic peptide secretion by the heart. Annu Rev Physiol 61, Sudoh T, Minamino N, Kangawa K & Matsuo H (1988) Brain natriuretic peptide-32: N-terminal six amino acid extended form of brain natriuretic peptide FEBS Journal 278 (2011) ª 2011 The Author Journal compilation ª 2011 FEBS 1801

11 Update on functional aspects of GC-A NPRA K. N. Pandey identified in porcine brain. Biochem Biophys Res Commun 155, Seilhamer JJ, Arfsten A, Miller JA, Lundquist P, Scarborough RM, Lewicki JA & Porter JG (1989) Human and canine gene homologs of porcine brain natriuretic peptide. Biochem Biophys Res Commun 165, Mukoyama M, Nakao K, Hosoda K, Suga S, Saito Y, Ogawa Y, Shirakami G, Jougasaki M, Obata K, Yasue H et al. (1991) Brain natriuretic peptide as a novel cardiac hormone in humans. Evidence for an exquisite dual natriuretic peptide system, atrial natriuretic peptide and brain natriuretic peptide. J Clin Invest 87, Vellaichamy E, Khurana ML, Fink J & Pandey KN (2005) Involvement of the NF-kappa B matrix metalloproteinase pathway in cardiac fibrosis of mice lacking guanylyl cyclase natriuretic peptide receptor A. J Biol Chem 280, Grepin C, Dagnino L, Robitaille L, Haberstroh L, Antakly T & Nemer M (1994) A hormone-encoding gene identifies a pathway for cardiac but not skeletal muscle gene transcription. Mol Cell Biol 14, Thuerauf DJ, Hanford DS & Glembotski CC (1994) Regulation of rat brain natriuretic peptide transcription. A potential role for GATA-related transcription factors in myocardial cell gene expression. J Biol Chem 269, Cameron V, Aitken G, Ellmers L, Kennedy M & Espiner E (1996) The sites of gene expression of atrial, brain, and C-type natriuretic peptides in mouse fetal development: temporal changes in embryos and placenta. Endocrinology 137, Ogawa Y, Nakao K, Nakagawa O, Komatsu Y, Hosoda K, Suga S, Arai H, Nagata K, Yoshida N & Imura H (1992) Human C-type natriuretic peptide. Characterization of the gene and peptide. Hypertension 19, Suga S, Nakao K, Itoh H, Komatsu Y, Ogawa Y, Hama N & Imura H (1992) Endothelial production of C-type natriuretic peptide and its marked augmentation by transforming growth factor-beta: possible existence of vascular natriuretic peptide system. J Clin Invest 90, Hagiwara H, Sakaguchi H, Itakura M, Yoshimoto T, Furuya M, Tanaka S & Hirose S (1994) Autocrine regulation of rat chondrocyte proliferation by natriuretic peptide C and its receptor, natriuretic peptide receptor-b. J Biol Chem 269, Wu C, Wu F, Pan J, Morser J & Wu Q (2003) Furin-mediated processing of Pro-C-type natriuretic peptide. J Biol Chem 278, Schulz-Knappe P, Forssmann K, Herbst F, Hock D, Pipkorn R & Forssmann WD (1988) Isolation and structural analysis of urodilatin, a new peptide of the cardiodilatin (ANP)-family extracted from human urine. Klin Wochenschr 66, Feller SM, Ma gert HJ, Schulz-Knappe P & Forssmann WG (1990) Urodilatin (hanf ) characteristics of a new atrial natriuretic factor peptide. In Atrial Natriuretic Factor (Struthers AD ed.), pp Blackwell, Oxford. 31 Saxenhofer H, Roselli A, Weidmann P, Forssmann WG, Bub A, Ferrari P & Shaw SG (1990) Urodilatin, a natriuretic factor from kidneys can modify renal and cardiovascular function in men. Am J Physiol 259, F832 F Schweitz H, Vigne P, Moinier D, Frelin CH & Lazdunski M (1992) A new member of the natriuretic peptide family is present in the venom of the green mamba (Dendroaspis angusticeps). J Biol Chem 267, Chinkers M, Garbers DL, Chang MS, Lowe DG, Chin HM, Goeddel DV & Schulz S (1989) A membrane form of guanylate cyclase is an atrial natriuretic peptide receptor. Nature 338, Schulz S, Singh S, Bellet RA, Singh G, Tubb DJ, Chin H & Garbers DL (1989) The primary structure of a plasma membrane guanylate cyclase demonstrates diversity within this new receptor family. Cell 58, Chang MS, Lowe DG, Lewis M, Hellmiss R, Chen E & Goeddel DV (1989) Differential activation by atrial and brain natriuretic peptides of two different receptor guanylate cyclases. Nature 341, Pandey KN & Singh S (1990) Molecular cloning and expression of murine guanylate cyclase atrial natriuretic factor receptor cdna. J Biol Chem 265, Garbers DL (1992) Guanylyl cyclase receptors and their endocrine, paracrine, and autocrine ligands. Cell 71, Koller KJ, de Sauvage FJ, Lowe DG & Goeddel DV (1992) Conservation of the kinaselike regulatory domain is essential for activation of the natriuretic peptide receptor guanylyl cyclases. Mol Cell Biol 12, Khurana ML & Pandey KN (1993) Receptor-mediated stimulatory effect of atrial natriuretic factor, brain natriuretic peptide, and C-type natriuretic peptide on testosterone production in purified mouse Leydig cells: activation of cholesterol side-chain cleavage enzyme. Endocrinology 133, Fuller F, Porter JG, Arfsten AE, Miller J, Schilling JW, Scarborough RM, Lewicki JA & Schenk DB (1988) Atrial natriuretic peptide clearance receptor. Complete sequence and functional expression of cdna clones. J Biol Chem 263, Koller KJ, Lowe DG, Bennett GL, Minamino N, Kangawa K, Matsuo H & Goeddel DV (1991) Selective 1802 FEBS Journal 278 (2011) ª 2011 The Author Journal compilation ª 2011 FEBS