Identification of a cell surface receptor common to germ and somatic cells.

The plasma membrane forms of guanylyl cyclase constitute a diverse family of cell surface receptors. An mRNA for the enzyme/receptor was first cloned from sea urchin testis after cross-linking studies suggested that guanylyl cyclase was a sperm receptor for egg peptides. The enzyme/receptor was shown to contain a single putative transmembrane domain, a large extracellular region that presumably binds peptide ligands, and an intracellular region that contains a protein kinase-like and a cyclase catalytic domain. The sea urchin cDNA was then used to isolate positive-hybridizing clones from mammalian tissues. At least two forms recognize natriuretic peptides and one form recognizes the heat-stable enterotoxins. In the case of the enterotoxin receptor, it remains to be shown whether or not an endogenous ligand exists that regulates enzyme activity. The discovery of this cell surface receptor family presents a new paradigm for second messenger signalling in that a low-molecular weight second messenger (cyclic GMP) is produced by the same protein that binds the extracellular ligand.     

Structural insights into the ligand binding domains of membrane bound guanylyl cyclases and natriuretic peptide receptors

Membrane bound guanylyl cyclases are single chain transmembrane receptors that produce the second messenger cGMP by either intra- or extracellular stimuli. This class of type I receptors contain an intracellular catalytic guanylyl cyclase domain, an adjacent kinase-like domain and an extracellular ligand binding domain though some receptors have their ligands yet to be identified. The most studied member is the atrial natriuretic peptide (ANP) receptor, which is involved in blood pressure regulation. Extracellular ANP binding induces a conformational change thereby activating the pre-oligomerized receptor leading to the production of cGMP. The recent crystal structure of the dimerized hormone binding domain of the ANP receptor provides a first three-dimensional view of this domain and can serve as a basis to structurally analyze mutagenesis, cross-linking, and genetic studies of this class of receptors as well as a non-catalytic homolog, the clearance receptor. The fold of the ligand binding domain is that of a bilobal periplasmic binding protein (PBP) very similar to that of the Leu/Ile/Val binding protein, AmiC, multi-domain transmembrane metabotropic glutamate receptors, and several DNA binding proteins such as the lactose repressor. Unlike these structural homologs, the guanylyl cyclase receptors bind much larger molecules at a site seemingly remote from the usual small molecule binding site in periplasmic binding protein folds. Detailed comparisons with these structural homologs offer insights into mechanisms of signal transduction and allosteric regulation, and into the remarkable usage of the periplasmic binding protein fold in multi-domain receptors/proteins.     

Guanylyl cyclase is a heat-stable enterotoxin receptor.

Plasma membrane forms of guanylyl cyclase have been shown to function as natriuretic peptide receptors. We describe a new clone (GC-C) encoding a guanylyl cyclase receptor for heat-stable enterotoxin. GC-C encodes a protein containing an extracellular amino acid sequence divergent from that of previously cloned guanylyl cyclases; however, the protein retains the intracellular protein kinase-like and cyclase catalytic domains. Expression of GC-C in COS-7 cells results in high guanylyl cyclase activity. In addition, heat-stable enterotoxin from E. coli, but not natriuretic peptides, causes marked elevations of cyclic GMP and is specifically bound by cells transfected with GC-C. The enterotoxin fails to elevate cyclic GMP in nontransfected cells or in cells transfected with the natriuretic peptide/guanylyl cyclase receptors. These results show that a heat-stable enterotoxin receptor responsible for acute diarrhea is a plasma membrane form of guanylyl cyclase.     

Human atrial natriuretic peptide receptor defines a new paradigm for second messenger signal transduction.

We isolated cDNAs encoding a 115 kd human atrial natriuretic peptide (alpha ANP) receptor (ANP-A receptor) that possesses guanylate cyclase activity, by low-stringency hybridization with sea urchin Arbacia punctulata membrane guanylate cyclase probes. The human ANP-A receptor has a 32 residue signal sequence followed by a 441 residue extracellular domain homologous to the 60 kd ANP-C receptor. A 21 residue transmembrane domain precedes a 568 residue cytoplasmic domain with homology to the protein kinase family and to a subunit of the soluble guanylate cyclase. COS-7 cells transfected with an ANP-A receptor expression vector displayed specific [125I]alpha ANP binding, and exhibited alpha ANP stimulated cGMP production. These data demonstrate a new paradigm of cellular signal transduction where extracellular ligand binding allosterically regulates cyclic nucleotide second-messenger production by a receptor cytoplasmic catalytic domain.     

Ligand-mediated endocytosis and intracellular sequestration of guanylyl cyclase/natriuretic peptide receptors: role of GDAY motif

The guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), also referred to as GC-A, is a single polypeptide molecule having a critical function in blood pressure regulation and cardiovascular homeostasis. GC-A/NPRA, which resides in the plasma membrane, consists of an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular cytoplasmic region containing a protein kinase-like homology domain (KHD) and a guanylyl cyclase (GC) catalytic domain. After binding with atrial and brain natriuretic peptides (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 through its lifetime. This review describes the roles of short-signal sequences in the internalization, trafficking, and intracellular redistribution of GC-A/NPRA from cell surface to cell interior. 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 disassociated ligands are degraded in the lysosomes. However, a small percentage of the ligand escapes the lysosomal degradative pathway, and is released intact into culture medium. Using pharmacologic and molecular perturbants, emphasis has been placed on the cellular regulation and processing of ligand-bound GC-A/NPRA in terms of receptor trafficking and down-regulation in intact cells. The discussion is concluded by examining the functions of short-signal sequence motifs in the cellular life-cycle of GC-A/NPRA, including endocytosis, trafficking, metabolic processing, inactivation, and/or down-regulation in model cell systems.     

The guanylyl cyclase-receptor family.

Guanylyl cyclase activity exists in the soluble and particulate fractions of most tissue homogenates. Recently, the mRNA encoding members of the enzyme family from both the soluble and particulate fraction have been cloned. The soluble form exists as a heterodimer, both subunits containing cyclase catalytic-like amino acid sequences. Both subunits must be expressed to observe enzyme activity. The particulate (plasma membrane) forms contain a single putative transmembrane domain that separates an extracellular, ligand-binding domain from an intracellular, protein kinase-like and cyclase catalytic region. The protein kinase-like domain appears to serve as an important regulatory element of the receptor.     

Internalization and trafficking of guanylyl cyclase/natriuretic peptide receptor-A

One of the principal loci involved in the regulatory action of atrial and brain natriuretic peptides (ANP and BNP) is guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), whose ligand-binding efficiency and GC catalytic activity vary remarkably in different target cells and tissues. In its mature form, NPRA resides in the plasma membrane and contains an extracellular ligand-binding domain, a single transmembrane region, and the intracellular protein kinase-like homology domain (KHD) and guanylyl cyclase (GC) catalytic domain. NPRA is a dynamic cellular macromolecule that traverses through different compartments of the cell through its lifetime. Binding of ligand to NPRA triggers a complex array of signal transduction events and accelerates the endocytosis. The endocytic transport is important in regulating signal transduction, formation of specialized signaling complexes, and modulation of specific components of internalization events. The present review describes the experiments which reveal the internalization of ligand-receptor complexes of NPRA, receptor trafficking and recycling, and delivery of both ligand-receptor molecules into subcellular compartments. The ligand-receptor complexes of NPRA are finally degraded within the lysosomes. The experimental evidence provides a consensus forum, which establishes the endocytosis, cellular trafficking, sequestration, and metabolic processing of ANP/NPRA complexes in the intact cells. The discussion is afforded to address the experimental insights into the mechanisms that cells utilize in modulating the delivery and metabolic processing of ligand-bound NPRA into the cell interior.     

Regulation of photoreceptor membrane guanylyl cyclases by guanylyl cyclase activator proteins

Guanylyl cyclase (GC) plays a central role in the responses of vertebrate rod and cone photoreceptors to light. cGMP is an internal messenger molecule of vertebrate phototransduction. Light stimulates hydrolysis of cGMP, causing the closure of cGMP-dependent cation channels in the plasma membranes of photoreceptor outer segments. Light also lowers the concentration of intracellular free Ca(2+) and by doing so it stimulates resynthesis of cGMP by guanylyl cyclase. The guanylyl cyclases that couple Ca(2+) to cGMP synthesis in photoreceptors are members of a family of transmembrane guanylyl cyclases that includes atrial natriuretic peptide receptors and the heat-stable enterotoxin receptor. The photoreceptor membrane guanylyl cyclases, RetGC-1 and RetGC-2 (also referred to as GC-E and GC-F), are regulated intracellularly by two Ca(2+)-binding proteins, GCAP-1 and GCAP-2. GCAPs bind Ca(2+) at three functional EF-hand structures. Several lines of biochemical evidence suggest that guanylyl cyclase activator proteins (GCAPs) bind constitutively to an intracellular domain of RetGCs. In the absence of Ca(2+) GCAP stimulates and in the presence of Ca(2+) it inhibits cyclase activity. Proper functioning of RetGC and GCAP is necessary not only for normal photoresponses but also for photoreceptor viability since mutations in RetGC and in GCAP cause photoreceptor degeneration.     

Dynamics of internalization and sequestration of guanylyl cyclase/atrial natriuretic peptide receptor-A

The guanylyl cyclase/natriuretic peptide receptor-A (NPRA), also referred to as GC-A, is a single polypeptide molecule. In its mature form, NPRA resides in the plasma membrane and consists of an extracellular ligand-binding domain, a single transmembrane-spanning region, and intracellular cytoplasmic domain that contains a protein kinase-like homology domain (KHD) and a guanylyl cyclase (GC) catalytic active site. The binding of atrial natriuretic peptide (ANP) to NPRA occurs at the plasma membrane; the receptor is synthesized on the polyribosomes of the endoplasmic reticulum, and is presumably degraded within the lysosomes. It is apparent that NPRA is a dynamic cellular macromolecule that traverses through different compartments of the cell through its lifetime. This review describes the experiments addressing the interaction of ANP with the NPRA, the receptor-mediated internalization and stoichiometric distribution of ANP-NPRA complexes from cell surface to cell interior, and its release into culture media. It is hypothesized that after internalization, the ligand-receptor complexes dissociate inside the cell and a population of NPRA recycles back to plasma membrane. Subsequently, some of the dissociated ligand molecules escape the lysosomal degradative pathway and are released intact into culture media, which reenter the cell by retroendocytotic mechanisms. By utilizing the pharmacologic and physiologic perturbants, the emphasis has been placed on the cellular regulation and processing of ligand-receptor complexes in intact cells. I conclude the discussion by examining the data available on the utilization of deletion mutations of NPRA cDNA, which has afforded experimental insights into the mechanisms the cell utilizes in modulating the expression and functioning of NPRA.     

17β-Estradiol Elevates cGMP and,via Plasma Membrane Recruitment of Protein Kinase GIα, Stimulates Ca2+ Efflux from Rat Hepatocytes

Rapid non-genomic effects of 17β-estradiol, the principal circulating estrogen, have been observed in a wide variety of cell types. Here we investigate rapid signaling effects of 17β-estradiol in rat hepatocytes. We show that, above a threshold concentration of 1 nm, 17β-estradiol, but not 17α-estradiol, stimulates particulate guanylyl cyclase to elevate cGMP, which through activation and plasma membrane recruitment of protein kinase G isoform Iα, stimulates plasma membrane Ca²⁺-ATPase-mediated Ca²⁺ efflux from rat hepatocytes. These effects are extremely rapid in onset and are mimicked by a membrane-impermeant 17β-estradiol-BSA conjugate, suggesting that 17β-estradiol acts at the extracellular face of the plasma membrane. We also show that 17β-estradiol binds specifically to the intact hepatocyte plasma membrane through an interaction that is competed by an excess of atrial natriuretic peptide but also shows many similarities to the pharmacological characteristics of the putative γ-adrenergic receptor. We, therefore, propose that the observed rapid signaling effects of 17β-estradiol are mediated either through the guanylyl cyclase A receptor for atrial natriuretic peptide or through the γ-adrenergic receptor, which is either itself a transmembrane guanylyl cyclase or activates a transmembrane guanylyl cyclase through cross-talk signaling.     

The membrane form of guanylate cyclase. Homology with a subunit of the cytoplasmic form of the enzyme

A cDNA clone for the membrane form of guanylate cyclase has been isolated from the testis of the sea urchin Strongylocentrotus purpuratus. An open reading frame predicts a protein of 1125 amino acids including an apparent signal peptide of 21 residues; a single transmembrane domain of 25 amino acids divided the mature protein into an amino-terminal, extracellular domain of 485 amino acids and a carboxyl domain of 594 intracellular amino acids. Three potential Asn-linked glycosylation sites were present in the proposed extracellular domain. The deduced protein sequence was homologous to the protein kinase family and contained limited but significant regions of identity with a low molecular weight atrial natriuretic peptide receptor. The carboxyl region (202 amino acids) was 42% identical with a subunit of the cytoplasmic form of guanylate cyclase recently cloned from bovine lung (Koesling, D., Herz, J., Gausepohl, H., Niroomand, F., Hinsch, K.-D., Mulsch, A., Bohme, E., Schultz, G., and Frank, R. (1988) FEBS Lett. 239, 29-34). Therefore, the membrane form of guanylate cyclase is a member of an apparently large family of proteins that includes the low molecular weight atrial natriuretic peptide receptor, the soluble form of guanylate cyclase and protein kinases.     

The guanylate cyclase/receptor family of proteins

Guanylate cyclase, which catalyzes the formation of cGMP from GTP, exists in both the soluble and particulate fractions of cells. At least two different cellular compartments for the particulate enzyme exist: the plasma membrane and cytoskeleton. The enzyme form found in the soluble fraction is a heterodimer that can be regulated by free radicals and nitrovasodilators, whereas the membrane form exists as a single-chain polypeptide that can be regulated by various peptides. These peptides include resact and speract obtained from eggs and atrial natriuretic peptides (ANP). The species of guanylate cyclase present in cytoskeletal fractions resists solubilization with non-ionic detergents; its structural properties are not yet known. cDNAs encoding the membrane form of guanylate cyclase have been isolated from different tissues and species, and in all cases the DNA sequences predict a protein containing a single transmembrane domain. The carboxyl (intracellular) domain is highly conserved from sea urchins through mammals, whereas the extracellular domain (amino terminus) varies considerably. The predicted amino acid sequences demonstrate that the membrane form of guanylate cyclase is a member of a diverse and complex family of proteins that includes a low molecular weight ANP receptor, protein kinases, and the cytoplasmic form of guanylate cyclase. cDNA encoding a membrane form of the enzyme from mammalian tissues has been expressed in cultured cells, and the expressed guanylate cyclase specifically binds ANP and is activated by ANP. The membrane form of guanylate cyclase, then, serves as a cell surface receptor, representing the first recognized protein to directly catalyze formation of a low molecular weight second messenger in response to ligand binding.     

The atrial natriuretic peptide receptor (NPR-A/GC-A) is dephosphorylated by distinct microcystin-sensitive and magnesium-dependent protein phosphatases

Natriuretic peptide receptor (NPR)-A is the primary signaling receptor for atrial natriuretic peptide and brain natriuretic peptide. Ligand binding to NPR-A rapidly activates its guanylyl cyclase domain, but its rate of cGMP synthesis declines with time. This waning of activity is called homologous desensitization and is mediated in part by receptor dephosphorylation. Here, we characterize two distinct NPR-A phosphatase activities. The serine/threonine protein phosphatase inhibitor, microcystin, inhibited the desensitization of NPR-A in membrane guanylyl cyclase assays in the absence of magnesium. EDTA also inhibited the desensitization, whereas MgCl(2) stimulated the desensitization. Because the effects of microcystin and EDTA were additive, and microcystin did not block the magnesium-dependent desensitization, the targets for these agents appear to be distinct. Incubation of membranes at 37 degrees C stimulated the dephosphorylation of NPR-A, and microcystin blocked the temperature-dependent dephosphorylation. The addition of MgCl(2) or MnCl(2), but not CaCl(2), further stimulated the dephosphorylation of NPR-A, and microcystin failed to inhibit this process. The desensitization required changes in the phosphorylation state of NPR-A because the guanylyl cyclase activity of a receptor variant containing glutamate substitutions at all six phosphorylation sites was unaffected by MgCl(2), EDTA, or microcystin. Together, these data indicate that NPR-A is regulated by two distinct phosphatases, possibly including a member of the protein phosphatase 2C family. Finally, we observed that the desensitization of NPR-A in membranes from mouse kidneys and NIH3T3 cells was increased by prior exposure to atrial natriuretic peptide, suggesting that hormone binding enhances receptor dephosphorylation.     

ANP stimulates hepatocyte Ca2+ efflux via plasma membrane recruitment of PKGIalpha

In rat hepatocytes, atrial natriuretic peptide (ANP) elevates cGMP through activation of particulate guanylyl cyclase and attenuates Ca²⁺ signals by stimulating net plasma membrane Ca²⁺ efflux. We show here that ANP-stimulated hepatocyte Ca²⁺ efflux is mediated by protein kinase G (PKG) isotype I. Furthermore, we show that ANP recruits endogenous PKGIα, but not PKGIβ, to the plasma membrane. These effects are mimicked by 8-bromo-cGMP, but not by the soluble guanylyl cyclase activators, sodium nitroprusside and YC-1. We propose that ANP, through localized cGMP elevation, promotes plasma membrane recruitment of PKGIα, which, in turn, stimulates Ca²⁺ efflux.     

Natriuretic peptide receptor-C signaling and regulation

The natriuretic peptides (NP) are a family of three polypeptide hormones termed atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). ANP regulates a variety of physiological parameters by interacting with its receptors present on the plasma membrane. These are of three subtypes NPR-A, NPR-B, and NPR-C. NPR-A and NPR-B are guanylyl cyclase receptors, whereas NPR-C is non-guanylyl cyclase receptor and is coupled to adenylyl cyclase inhibition or phospholipase C activation through inhibitory guanine nucleotide regulatory protein (Gi). ANP, BNP, CNP, as well as C-ANP(4-23), a ring deleted peptide that specifically interacts with NPR-C receptor inhibit adenylyl cyclase activity through Gi protein. Unlike other G-protein-coupled receptors, NPR-C receptors have a single transmembrane domain and a short cytoplasmic domain of 37 amino acids, which has a structural specificity like those of other single transmembrane domain receptors. A 37 amino acid cytoplasmic peptide is sufficient to inhibit adenylyl cyclase activity with an apparent Ki similar to that of ANP(99-126) or C-ANP(4-23). In addition, C-ANP(4-23) also stimulates phosphatidyl inositol (PI) turnover in vascular smooth muscle cells (VSMC) which is attenuated by dbcAMP and cAMP-stimulatory agonists, suggesting that NPR-C receptor-mediated inhibition of adenylyl cyclase and resultant decreased levels of cAMP may be responsible for NPR-C-mediated stimulation of PI turnover. Furthermore, the activation of NPR-C receptor by C-ANP(4-23) and CNP inhibits the mitogen-activated protein kinase activity stimulated by endothelin-3, platelet-derived growth factor, phorbol-12 myristate 13-acetate, suggesting that NPR-C receptor might also be coupled to other signal transduction system or that there may be an interaction of the NPR-C receptor and some other signaling pathways. In this review article, NPR-C receptor coupling to different signaling pathways and their regulation will be discussed.     

Intracellular trafficking and metabolic turnover ofligand-bound guanylyl cyclase/atrial natriuretic peptide receptor-A into subcellular compartments

Atrial natriuretic peptide (ANP) is the first described member of the natriuretic peptide hormone family. ANP elicits natriuretic, diuretic, vasorelaxant and antiproliferative effects, important factors in the control of blood pressure homeostasis. One of the principal loci involved in the regulatory action of ANP is the guanylyl cyclase-linked ANP-receptor which has been designated as NPRA, also referred to as GC-A, whose ANP-binding efficiency and guanylyl cyclase activity vary remarkably in different target tissues. However, the cellular and molecular basis of these activities and the functional expression and regulation of NPRA are not well understood. The mature form of receptor resides in the plasma membrane and consists of an extracellular ligand-binding domain, a single transmembrane-spanning region, and intracellular protein kinase-like homology and guanylyl cyclase catalytic domains. In this review, emphasis has been placed on the interaction of ANP with NPRA, the ligand-mediated endocytosis, trafficking, and subcellular distribution of ligand-receptor complexes from cell surface to the intracellular compartments. Furthermore, it is implicated that after internalization, the ANP/NPRA complexes dissociate into the subcellular compartments and a population of receptor recycles back to the plasma membrane. This is an interesting area of research in the natriuretic peptide receptor field because there is currently debate over whether ANP/NPRA complexes internalize at all or whether cell utilizes some other mechanisms to release ANP from the bound receptor molecules. Indeed, controversy exist since it has been previously reported by default that among the three natriuretic peptide receptors only NPRC internalizes with bound ligand. Hence, from a thematic standpoint it is clearly evident that there is a current need to review this subject and provide a consensus forum that establishes the cellular trafficking, sequestration and processing of ANP/NPRA complexes in intact cells. Towards this aim the cellular life-cycle of NPRA will be described in the context of ANP-binding, internalization, metabolic processing, and/or inactivation, down-regulation, and degradation of ligand-receptor complexes in model cell systems.     

Primary structure and functional expression of the human receptor for Escherichia coli heat-stable enterotoxin.

Heat-stable enterotoxin (STa) produced by Escherichia coli induces intestinal secretion in mammals by binding to the brush border membrane of the small intestine and activating guanylyl cyclase. We report here the cloning and expression of a cDNA encoding the human receptor for STa. The receptor contains both an extracellular ligand binding site and a cytoplasmic guanylyl cyclase catalytic domain, making it a member of the same receptor family as the natriuretic peptide receptors. Stable mammalian cell lines over-expressing the STa receptor specifically bind 125I-STa (Kd approximately 1.0 nM) and respond to STa by dramatically increasing (approximately 50-fold) cellular cGMP levels. Sequence comparisons between the human and the rat STa receptors show less conservation in the extracellular domain than similar comparisons of natriuretic peptide receptors. This divergence may indicate important species differences in ligand-receptor interaction.     

Atrial natriuretic peptide induces natriuretic peptide receptor-cGMP-dependent protein kinase interaction

Circulating natriuretic peptides such as atrial natriuretic peptide (ANP) counterbalance the effects of hypertension and inhibit cardiac hypertrophy by activating cGMP-dependent protein kinase (PKG). Natriuretic peptide binding to type I receptors (NPRA and NPRB) activates their intrinsic guanylyl cyclase activity, resulting in a rapid increase in cytosolic cGMP that subsequently activates PKG. Phosphorylation of the receptor by an unknown serine/threonine kinase is required before ligand binding can activate the cyclase. While searching for downstream PKG partners using a yeast two-hybrid screen of a human heart cDNA library, we unexpectedly found an upstream association with NPRA. PKG is a serine/threonine kinase capable of phosphorylating NPRA in vitro; however, regulation of NPRA by PKG has not been previously reported. Here we show that PKG is recruited to the plasma membrane following ANP treatment, an effect that can be blocked by pharmacological inhibition of PKG activation. Furthermore, PKG participates in a ligand-dependent gain-of-function loop that significantly increases the intrinsic cyclase activity of the receptor. PKG translocation is ANP-dependent but not nitric oxide-dependent. Our results suggest that anchoring of PKG to NPRA is a key event after ligand binding that determines distal effects. As such, the NPRA-PKG association may represent a novel mechanism for compartmentation of cGMP-mediated signaling and regulation of receptor sensitivity.     

C-type natriuretic peptide and guanylyl cyclase B receptor

Guanylyl cyclases (GC) are widely distributed enzymes that signal via the production of the second messenger cGMP. The particulate guanylyl cyclases share a similar topology: an extracellular ligand binding domain and intracellular regulatory kinase-homology and cyclase catalytic domains. The natriuretic peptide receptors GC-A and -B mediate the effects of a family of peptides, atrial, B- and C-type natriuretic peptide (ANP, BNP and CNP, respectively), with natriuretic, diuretic and vasorelaxant properties. ANP and BNP, through the activation of GC-A, act as endocrine hormones to regulate blood pressure and volume, and inhibit cardiac hypertrophy. CNP, on the other hand, acts in an autocrine/paracrine fashion to induce vasorelaxation and vascular remodeling, and to regulate bone growth through its cognate receptor GC-B. GC-B, like GC-A, is phosphorylated in the basal state, and undergoes both homologous and heterologous desensitization, reflected by dephosphorylation of specific sites in the kinase-homology domain. This review will examine the structure and function of GC-B, and summarize the physiological processes in which this receptor is thought to participate.     

A disulfide-bridged mutant of natriuretic peptide receptor-A displays constitutive activity. Role of receptor dimerization in signal transduction

Natriuretic peptide receptor-A (NPR-A), a particulate guanylyl cyclase receptor, is composed of an extracellular domain (ECD) with a ligand binding site, a transmembrane spanning, a kinase homology domain (KHD), and a guanylyl cyclase domain. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), the natural agonists, bind and activate the receptor leading to cyclic GMP production. This receptor has been reported to be spontaneously dimeric or oligomeric. In response to agonists, the KHD-mediated guanylate cyclase repression is removed, and it is assumed that ATP binds to the KHD. Since NPR-A displays a pair of juxtamembrane cysteines separated by 8 residues, we hypothesized that the removal of one of those cysteines would leave the other unpaired and reactive, thus susceptible to form an interchain disulfide bridge and to favor the dimeric interactions. Here we show that NPR-AC423S mutant, expressed mainly as a covalent dimer, increases the affinity of pBNP for this receptor by enhancing a high affinity binding component. Dimerization primarily depends on ECD since a secreted NPR-A C423S soluble ectodomain (ECDC423S) also documents a covalent dimer. ANP binding to the unmutated ECD yields up to 80-fold affinity loss as compared with the membrane receptor. However, the ECD C423S mutation restores a high binding affinity. Furthermore, C423S mutation leads to cellular constitutive activation (20-40-fold) of basal catalytic production of cyclic GMP by the full-length mutant. In vitro particulate guanylyl cyclase assays demonstrate that NPR-AC423S displays an increased sensitivity to ATP treatment alone and that the effect of ANP + ATP joint treatment is cumulative instead of synergistic. Finally, the cellular and particulate guanylyl cyclase assays indicate that the receptor is desensitized to agonist stimulation. We conclude the following: 1) dimers are functional units of NPR-A guanylyl cyclase activation; and 2) agonists are inducing dimeric contact of the juxtamembranous region leading to the removal of the KHD-mediated guanylyl cyclase repression, hence allowing catalytic activation.