Determination of the binding site on the extracellular domain of guanylyl cyclase C to heat-stable enterotoxin.

Guanylyl cyclase C, one of the family of membrane-bound guanylyl cyclases, consists of an extracellular domain and an intracellular domain, which are connected by a single transmembrane polypeptide. The extracellular domain binds unique small polypeptides with high specificity, which include the endogenous peptide hormones, guanylin and uroguanylin, as well as an exogenous enterotoxigenic peptide, heat-stable enterotoxin, secreted by pathogenic Escherichia coli. Information on this specific binding is propagated into the intracellular domain, followed by the synthesis of cGMP, a second messenger that regulates a variety of intracellular physiological processes. This study reports the design of a photoaffinity labeled analog of heat-stable enterotoxin (biotinyl-(AC(5))(2)-[Gly(4), Pap(11)]STp(4-17)), which incorporates a Pap residue (p-azidophenylalanine) at position 11 and a biotin moiety at the N terminus, and the use of this analog to determine the ligand-binding region of the extracellular domain of guanylyl cyclase C. The endoproteinase Lys-C digestion of the extracellular domain, which was covalently labeled by this ligand, and mass spectrometric analyses of the digest revealed that the ligand specifically binds to the region (residue 387 to residue 393) of guanylyl cyclase C. This region is localized close to the transmembrane portion of guanylyl cyclase C on the external cellular surface. This result was further confirmed by characterization of site-directed mutants of guanylyl cyclase C in which each amino acid residue was substituted by an Ala residue instead of residues normally located in the region. This experiment provides the first direct demonstration of the ligand-binding site of guanylyl cyclase C and will contribute toward an understanding of the receptor recognition of a ligand and the modeling of the interaction of the receptor and its ligand at the molecular level.     

Instead of binding calcium, one of the EF-hand structures in guanylyl cyclase activating protein-2 is required for targeting photoreceptor guanylyl cyclase

Guanylyl cyclase activator proteins (GCAPs) are calcium-binding proteins closely related to recoverin, neurocalcin, and many other neuronal Ca(2+)-sensor proteins of the EF-hand superfamily. GCAP-1 and GCAP-2 interact with the intracellular portion of photoreceptor membrane guanylyl cyclase and stimulate its activity by promoting tight dimerization of the cyclase subunits. At low free Ca(2+) concentrations, the activator form of GCAP-2 associates into a dimer, which dissociates when GCAP-2 binds Ca(2+) and becomes inhibitor of the cyclase. GCAP-2 is known to have three active EF-hands and one additional EF-hand-like structure, EF-1, that deviates form the EF-hand consensus sequence. We have found that various point mutations within the EF-1 domain can specifically affect the ability of GCAP-2 to interact with the target cyclase but do not hamper the ability of GCAP-2 to undergo reversible Ca(2+)-sensitive dimerization. Point mutations within the EF-1 region can interfere with both the activation of the cyclase by the Ca(2+)-free form of GCAP-2 and the inhibition of retGC basal activity by the Ca(2+)-loaded GCAP-2. Our results strongly indicate that evolutionary conserved and GCAP-specific amino acid residues within the EF-1 can create a contact surface for binding GCAP-2 to the cyclase. Apparently, in the course of evolution GCAP-2 exchanged the ability of its first EF-hand motif to bind Ca(2+) for the ability to interact with the target enzyme.     

Identification of a binding region on Escherichia coli heat-stable enterotoxin to intestinal guanylyl cyclase C

The heat-stable enterotoxin (ST), produced by Escherichia coli, causes acute diarrhea in infants and domestic animals by activation of the intestinal membrane-bound receptor, guanylyl cyclase C. We have investigated a region on the ST molecule, which is recognized by the receptor, by introducing a photochromophore, p-azidophenylalanine (Pap), into three different regions of STp(4–17), which has the full toxic activity. Each ST analog bound to the receptor, but only STp(4–17) containing a Pap residue at position 11 in the central portion of the ST molecule, showed a high efficiency in the reaction which cross- linked with the receptor by UV radiation. These data clearly demonstrate that the region of the ST molecule encompassing the Asn11 residue directly interacts with the receptor.     

Identification of a binding region on Escherichia coli heat-stable enterotoxin to intestinal guanylyl cyclase C

Summary The heat-stable enterotoxin (ST), produced byEscherichia coli, causes acute diarrhea in infants and domestic animals by activation of the intestinal membrane-bound receptor, guanylyl cyclase C. We have investigated a region on the ST molecule, which is recognized by the receptor, by introducing a photochromophore,p-azidophenylalanine (Pap), into three different regions of STp(4–17), which has the full toxic activity. Each ST analog bound to the receptor, but only STp(4–17) containing a Pap residue at position 11 in the central portion of the ST molecule, showed a high efficiency in the reaction which cross-linked with the receptor by UV radiation. These data clearly demonstrate that the region of the ST molecule encompassing the Asn¹¹ residue directly interacts with the receptor.     

Human natriuretic peptide receptor-A guanylyl cyclase is self-associated prior to hormone binding.

The human natriuretic peptide receptor-A (NPR-A) guanylyl cyclase is specifically activated to synthesize cGMP by binding of atrial natriuretic peptide (ANP) to the receptor's extracellular domain. In this report, NPR-A monoclonal and polyclonal antibodies were used to assess the aggregation status of wild-type NPR-A and a truncation mutant lacking most of the NPR-A cytoplasmic domain. On intact human embryonic kidney 293 cells, in the absence of ANP, recombinant human NPR-A is self-aggregated through disulfide bonds in an M(r) > 500,000, possibly tetrameric, complex. Under nonreducing conditions, truncated NPR-A was a monomer, indicating that the cytoplasmic domain is necessary for NPR-A self-association. In the presence of the homobifunctional cross-linker dithiobis(succinimidyl propionate), or disuccimidyl suberate, truncated NPR-A could be cross-linked as a dimer and trimer only in the presence of ANP. Wild-type NPR-A was cross-linked with disuccinimidyl suberate to an M(r) > 500,000 species in the absence of ANP, and with ANP, a smaller, M(r) approximately 400,000 receptor trimer cross-linking product was observed, together with the larger, possibly tetrameric complex. When whole cell stimulation of cGMP production by ANP was tested on the low level of endogenous 293 cell NPR-A, maximal stimulation was observed regardless of truncated NPR-A overexpression. The absence of a dominant negative effect by the truncated NPR-A, together with the cross-linking data, demonstrates that preassociated NPR-A is the functionally relevant form of this receptor.     

Tyrosine phosphorylation of the human guanylyl cyclase C receptor

Tyrosine phosphorylation events are key components of several cellular signal transduction pathways. This study describes a novel method for identification of substrates for tyrosine kinases. Co-expression of the tyrosine kinase EphB1 with the intracellular domain of guanylyl cyclase C (GCC) inEscherichia coli cells resulted in tyrosine phosphorylation of GCC, indicating that GCC is a potential substrate for tyrosine kinases. Indeed, GCC expressed in mammalian cells is tyrosine phosphorylated, suggesting that tyrosine phosphorylation may play a role in regulation of GCC signalling. This is the first demonstration of tyrosine phosphorylation of any member of the family of membrane-associated guanylyl cyclases.     

Mechanism of binding of NO to soluble guanylyl cyclase: implication for the second NO binding to the heme proximal site

Soluble guanylyl cyclase (sGC), the key enzyme for the formation of second messenger cyclic GMP, is an authentic sensor for nitric oxide (NO). Binding of NO to sGC leads to strong activation of the enzyme activity. Multiple molecules and steps of binding of NO to sGC have been implicated, but the target of the second NO and the detailed binding mechanism remain controversial. In this study, we used (15)NO and (14)NO and anaerobic sequential mixing-freeze-quench electron paramagnetic resonance to unambiguously confirm that the heme Fe is the target of the second NO. The linear dependence on NO concentration up to 600 s(-1) for the observed rate of the second step of NO binding not only indicates that the binding site of the second NO is different from that in the first step, i.e., the proximal site of the heme, but also supports a concerted mechanism in which the dissociation of the His105 proximal ligand occurs simultaneously with the binding of the second NO molecule. Computer modeling successfully predicts the kinetics of formation of a set of five-coordinate NO complexes with the ligand on either the distal or proximal site and supports the selective release of NO from the distal side of the transient bis-NO-sGC complex. Thus, as has been demonstrated with cytochrome c', a five-coordinate NO-sGC complex containing a proximal NO is formed after the binding of the second NO.     

Topological mimicry and epitope duplication in the guanylyl cyclase C receptor.

Guanylyl cyclase C (GCC) is the receptor for the gastrointestinal hormones, guanylin, and uroguanylin, in addition to the bacterial heat-stable enterotoxins, which are one of the major causes of watery diarrhea the world over. GCC is expressed in intestinal cells, colorectal tumor tissue and tumors originating from metastasis of the colorectal carcinoma. We have earlier generated a monoclonal antibody to human GCC, GCC:B10, which was useful for the immunohistochemical localization of the receptor in the rat intestine (Nandi A et al., 1997, J Cell Biochem 66:500-511), and identified its epitope to a 63-amino acid stretch in the intracellular domain of GCC. In view of the potential that this antibody has for the identification of colorectal tumors, we have characterized the epitope for GCC:B10 in this study. Overlapping peptide synthesis indicated that the epitope was contained in the sequence HIPPENIFPLE. This sequence was unique to GCC, and despite a short stretch of homology with serum amyloid protein and pertussis toxin, no cross reactivity was detected. The core epitope was delineated using a random hexameric phage display library, and two categories of sequences were identified, containing either a single, or two adjacent proline residues. No sequence identified by phage display was identical to the epitope present in GCC, indicating that phage sequences represented mimotopes of the native epitope. Alignment of these sequences with HIPPENIFPLE suggested duplication of the recognition motif, which was confirmed by peptide synthesis. These studies allowed us not only to define the requirements of epitope recognition by GCC:B10 monoclonal antibody, but also to describe a novel means of epitope recognition involving topological mimicry and probable duplication of the cognate epitope in the native guanylyl cyclase C receptor sequence.     

Three-dimensional structure of guanylyl cyclase activating protein-2, a calcium-sensitive modulator of photoreceptor guanylyl cyclases.

Guanylyl cyclase activating protein-2 (GCAP-2) is a Ca2+-sensitive regulator of phototransduction in retinal photoreceptor cells. GCAP-2 activates retinal guanylyl cyclases at low Ca2+ concentration (<100 nM) and inhibits them at high Ca2+ (>500 nM). The light-induced lowering of the Ca2+ level from approximately 500 nM in the dark to approximately 50 nM following illumination is known to play a key role in visual recovery and adaptation. We report here the three-dimensional structure of unmyristoylated GCAP-2 with three bound Ca2+ ions as determined by nuclear magnetic resonance spectroscopy of recombinant, isotopically labeled protein. GCAP-2 contains four EF-hand motifs arranged in a compact tandem array like that seen previously in recoverin. The root mean square deviation of the main chain atoms in the EF-hand regions is 2.2 A in comparing the Ca2+-bound structures of GCAP-2 and recoverin. EF-1, as in recoverin, does not bind calcium because it contains a disabling Cys-Pro sequence. GCAP-2 differs from recoverin in that the calcium ion binds to EF-4 in addition to EF-2 and EF-3. A prominent exposed patch of hydrophobic residues formed by EF-1 and EF-2 (Leu24, Trp27, Phe31, Phe45, Phe48, Phe49, Tyr81, Val82, Leu85, and Leu89) may serve as a target-binding site for the transmission of calcium signals to guanylyl cyclase.     

Cloning and functional expression of the rat alpha(2) subunit of soluble guanylyl cyclase

Nitric oxide-sensitive guanylyl cyclase is a heterodimeric enzyme consisting of one alpha and one beta subunit. Here, we clone the first alpha(2) subunit ortholog and functionally express the cDNA in Sf-9 cells. Our data indicate a high degree of conservation of the primary sequence and functional activity of the rat alpha(2) subunit.     

Identification of critical cis elements involved in mediating Epstein-Barr virus nuclear antigen 2-dependent activity of an enhancer located upstream of the viral BamHI C promoter.

The six genes encoding the Epstein-Barr virus nuclear antigens (EBNAs) are transcribed from one of two promoters, BamHI C promoter (Cp) or BamHI W promoter (Wp), located near the left end of the viral genome. During the establishment of viral latency in B lymphocytes, Wp is used exclusively before a switch to Cp usage. We and others have previously identified an enhancer in the region upstream of Cp which requires EBNA 2 for activity (M. Woisetschlaeger, X. W. Jin, C. N. Yandava, L. A. Furmanski, J. L. Strominger, and S. H. Speck, Proc. Natl. Acad. Sci. USA 88:3942-3946, 1991; N. S. Sung, S. Kenney, D. Gutsch, and J. S. Pagano, J. Virol. 65:2164-2169, 1991). Infection of B lymphocytes with a mutant virus lacking the EBNA 2 gene results in prolonged usage of Wp and failure to switch to Cp usage, indicating that EBNA 2 transactivation of the enhancer upstream of Cp may be critical for promoter switching. In this study, we have defined the minimal EBNA 2-dependent enhancer by using a series of deletion mutants. The results of site-directed mutagenesis revealed that there are three regions of the enhancer that are important for activity, two of which appear to bind B-lymphocyte-specific factors.