Title: The Linker Region in Receptor Guanylyl Cyclases Is a Key Regulatory Module
Abstract: Receptor guanylyl cyclases are multidomain proteins, and ligand binding to the extracellular domain increases the levels of intracellular cGMP. The intracellular domain of these receptors is composed of a kinase homology domain (KHD), a linker of ∼70 amino acids, followed by the C-terminal guanylyl cyclase domain. Mechanisms by which these receptors are allosterically regulated by ligand binding to the extracellular domain and ATP binding to the KHD are not completely understood. Here we examine the role of the linker region in receptor guanylyl cyclases by a series of point mutations in receptor guanylyl cyclase C. The linker region is predicted to adopt a coiled coil structure and aid in dimerization, but we find that the effects of mutations neither follow a pattern predicted for a coiled coil peptide nor abrogate dimerization. Importantly, this region is critical for repressing the guanylyl cyclase activity of the receptor in the absence of ligand and permitting ligand-mediated activation of the cyclase domain. Mutant receptors with high basal guanylyl cyclase activity show no further activation in the presence of non-ionic detergents, suggesting that hydrophobic interactions in the basal and inactive conformation of the guanylyl cyclase domain are disrupted by mutation. Equivalent mutations in the linker region of guanylyl cyclase A also elevated the basal activity and abolished ligand- and detergent-mediated activation. We, therefore, have defined a key regulatory role for the linker region of receptor guanylyl cyclases which serves as a transducer of information from the extracellular domain via the KHD to the catalytic domain. Receptor guanylyl cyclases are multidomain proteins, and ligand binding to the extracellular domain increases the levels of intracellular cGMP. The intracellular domain of these receptors is composed of a kinase homology domain (KHD), a linker of ∼70 amino acids, followed by the C-terminal guanylyl cyclase domain. Mechanisms by which these receptors are allosterically regulated by ligand binding to the extracellular domain and ATP binding to the KHD are not completely understood. Here we examine the role of the linker region in receptor guanylyl cyclases by a series of point mutations in receptor guanylyl cyclase C. The linker region is predicted to adopt a coiled coil structure and aid in dimerization, but we find that the effects of mutations neither follow a pattern predicted for a coiled coil peptide nor abrogate dimerization. Importantly, this region is critical for repressing the guanylyl cyclase activity of the receptor in the absence of ligand and permitting ligand-mediated activation of the cyclase domain. Mutant receptors with high basal guanylyl cyclase activity show no further activation in the presence of non-ionic detergents, suggesting that hydrophobic interactions in the basal and inactive conformation of the guanylyl cyclase domain are disrupted by mutation. Equivalent mutations in the linker region of guanylyl cyclase A also elevated the basal activity and abolished ligand- and detergent-mediated activation. We, therefore, have defined a key regulatory role for the linker region of receptor guanylyl cyclases which serves as a transducer of information from the extracellular domain via the KHD to the catalytic domain. In transmembrane receptors a series of conformational changes are required to transmit the information of ligand binding (an extracellular signal) to the interior of the cell, resulting in either altered interaction with signaling intermediates or in the regulation of a catalytic activity present in the receptor. In these multidomain receptors, where the ligand binding and effector domains are present in the same polypeptide chain, the relay of conformational changes is under the exquisite control of post-translational modifications or precise structural alterations.Receptor guanylyl cyclases (GCs) 4The abbreviations used are: GCguanylyl cyclaseGC-Aguanylyl cyclase AGC-Cguanylyl cyclase C (heat-stable enterotoxin receptor)GCAP-1GC-activating protein 1GSTglutathione S-transferaseANPatrial natriuretic peptideKHDkinase homology domainRetGC-1retinal guanylyl cyclaseSTheat-stable enterotoxinHAMPhistidine kinases, adenylyl cyclases, methyl accepting chemotactic receptors, and phosphatases. 4The abbreviations used are: GCguanylyl cyclaseGC-Aguanylyl cyclase AGC-Cguanylyl cyclase C (heat-stable enterotoxin receptor)GCAP-1GC-activating protein 1GSTglutathione S-transferaseANPatrial natriuretic peptideKHDkinase homology domainRetGC-1retinal guanylyl cyclaseSTheat-stable enterotoxinHAMPhistidine kinases, adenylyl cyclases, methyl accepting chemotactic receptors, and phosphatases. have an N-terminal extracellular ligand binding domain, a single transmembrane domain, and a C-terminal intracellular domain (1Padayatti P.S. Pattanaik P. Ma X. van den Akker F. Pharmacol. Ther. 2004; 104: 83-99Crossref PubMed Scopus (46) Google Scholar). Binding of ligands to the extracellular domain elicits a conformational change that increases the guanylyl cyclase activity of the receptor, resulting in increased cGMP production. The intracellular domain of receptor GCs contains a region that shares considerable sequence similarity to protein kinases and is referred to as the kinase homology domain (KHD). Binding of ATP to the KHD induces a conformational change that regulates cGMP production by the guanylyl cyclase domain (2Jaleel M. Saha S. Shenoy A.R. Visweswariah S.S. Biochemistry. 2006; 45: 1888-1898Crossref PubMed Scopus (26) Google Scholar). Thus, receptor GCs exemplify the intricate interactions between domains in transducing the signal from an extracellular ligand to the interior of the cell.The amino acid sequences of the extracellular domain of mammalian receptor GCs vary (less than ∼15% similarity), as would be expected given the diversity in the ligands that bind to and activate these receptors. The KHD shows ∼25–30% conservation in amino acid sequence across receptor GCs, and computational modeling has not only suggested that this region could adopt the overall structure of a protein kinase but also identified specific residues that could interact with ATP (2Jaleel M. Saha S. Shenoy A.R. Visweswariah S.S. Biochemistry. 2006; 45: 1888-1898Crossref PubMed Scopus (26) Google Scholar, 3Bhandari R. Srinivasan N. Mahaboobi M. Ghanekar Y. Suguna K. Visweswariah S.S. Biochemistry. 2001; 40: 9196-9206Crossref PubMed Scopus (30) Google Scholar). The catalytic domains of mammalian receptor GCs are more conserved (∼80% sequence similarity). The gradual increase in sequence similarity across the various domains, with the extracellular domain being the most diverse and the cyclase domains sharing the maximum sequence similarity, is a reflection of the ability of these receptor GCs to converge diverse extracellular signals to a unified output of cGMP production. The guanylyl cyclase domains of receptor GCs can be classified as members of the Class III family of nucleotide cyclases (4Linder J.U. Schultz J.E. Cell. Signal. 2003; 15: 1081-1089Crossref PubMed Scopus (142) Google Scholar). The recent crystal structures of a bacterial guanylyl cyclase (5Rauch A. Leipelt M. Russwurm M. Steegborn C. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 15720-15725Crossref PubMed Scopus (87) Google Scholar) and a eukaryotic soluble guanylyl cyclase (6Winger J.A. Derbyshire E.R. Lamers M.H. Marletta M.A. Kuriyan J. BMC Struct. Biol. 2008; 8: 42Crossref PubMed Scopus (79) Google Scholar) show similarities in the overall three-dimensional structure of adenylyl and guanylyl cyclases and also highlight the critical residues that determine substrate utilization (either ATP or GTP) in these enzymes.Guanylyl cyclase C (GC-C) serves as the receptor for the guanylin family of endogenous peptides as well as for the exogenous heat-stable enterotoxin (ST) peptides secreted by enterotoxigenic bacteria (7Forte L.R. Regul. Pept. 1999; 81: 25-39Crossref PubMed Scopus (115) Google Scholar, 8Vaandrager A.B. Mol. Cell. Biochem. 2002; 230: 73-83Crossref PubMed Scopus (111) Google Scholar). GC-C is predominantly expressed on the apical surface of epithelial cells in the intestine, although robust extra-intestinal expression is observed in the kidney and reproductive tissues of the rat (9Forte L.R. Krause W.J. Freeman R.H. Am. J. Physiol. 1989; 257: F874-F881PubMed Google Scholar, 10Jaleel M. London R.M. Eber S.L. Forte L.R. Visweswariah S.S. Biol. Reprod. 2002; 67: 1975-1980Crossref PubMed Scopus (30) Google Scholar, 11Nandi A. Bhandari R. Visweswariah S.S. J. Cell. Biochem. 1997; 66: 500-511Crossref PubMed Scopus (33) Google Scholar, 12Qian X. Prabhakar S. Nandi A. Visweswariah S.S. Goy M.F. Endocrinology. 2000; 141: 3210-3224Crossref PubMed Scopus (37) Google Scholar). The extracellular domain of GC-C is glycosylated, and we have shown the importance of glycosylation in regulating receptor desensitization in colonic cells. We have also identified a critical residue (Lys-516) in the KHD of GC-C as being important for KHD-mediated modulation of the guanylyl cyclase activity (2Jaleel M. Saha S. Shenoy A.R. Visweswariah S.S. Biochemistry. 2006; 45: 1888-1898Crossref PubMed Scopus (26) Google Scholar, 3Bhandari R. Srinivasan N. Mahaboobi M. Ghanekar Y. Suguna K. Visweswariah S.S. Biochemistry. 2001; 40: 9196-9206Crossref PubMed Scopus (30) Google Scholar).A sequence of ∼70 amino acids is found between the KHD and the guanylyl cyclase domain of receptor GCs, which we refer to here as the linker region (13Biswas K.H. Shenoy A.R. Dutta A. Visweswariah S.S. J. Mol. Evol. 2009; 68: 587-602Crossref PubMed Scopus (33) Google Scholar). This region is predicted to form an amphipathic α-helix and could also adopt a coiled coil conformation (14Hirayama T. Wada A. Hidaka Y. Fujisawa J. Takeda Y. Shimonishi Y. Microb. Pathog. 1993; 15: 283-291Crossref PubMed Scopus (15) Google Scholar, 15van den Akker F. Zhang X. Miyagi M. Huo X. Misono K.S. Yee V.C. Nature. 2000; 406: 101-104Crossref PubMed Scopus (138) Google Scholar). The linker region is also present in soluble (cytosolic) guanylyl cyclases where it connects the N-terminal heme binding regulatory domain to the C-terminal catalytic cyclase domain. The linker region is suggested to act as a dimerization module in receptor GCs (16Ramamurthy V. Tucker C. Wilkie S.E. Daggett V. Hunt D.M. Hurley J.B. J. Biol. Chem. 2001; 276: 26218-26229Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 17Vijayachandra K. Guruprasad M. Bhandari R. Manjunath U.H. Somesh B.P. Srinivasan N. Suguna K. Visweswariah S.S. Biochemistry. 2000; 39: 16075-16083Crossref PubMed Scopus (25) Google Scholar, 18Wilson E.M. Chinkers M. Biochemistry. 1995; 34: 4696-4701Crossref PubMed Scopus (151) Google Scholar) and has also been implicated in heterodimerization of the α and β subunits of soluble guanylyl cyclases (19Shiga T. Suzuki N. Zool. Sci. 2005; 22: 735-742Crossref PubMed Scopus (12) Google Scholar, 20Zhou Z. Gross S. Roussos C. Meurer S. Müller-Esterl W. Papapetropoulos A. J. Biol. Chem. 2004; 279: 24935-24943Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). However, there are several reports to the contrary that indicate that the linker does not affect the dimerization of receptor GCs (14Hirayama T. Wada A. Hidaka Y. Fujisawa J. Takeda Y. Shimonishi Y. Microb. Pathog. 1993; 15: 283-291Crossref PubMed Scopus (15) Google Scholar, 15van den Akker F. Zhang X. Miyagi M. Huo X. Misono K.S. Yee V.C. Nature. 2000; 406: 101-104Crossref PubMed Scopus (138) Google Scholar). Nevertheless, the critical importance of the linker in regulating the activity of receptor GCs is shown by the fact that mutations in this region of the retinal guanylyl cyclase (RetGC-1) are associated with autosomal dominant cone-rod dystrophy in humans (16Ramamurthy V. Tucker C. Wilkie S.E. Daggett V. Hunt D.M. Hurley J.B. J. Biol. Chem. 2001; 276: 26218-26229Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 21Smith M. Whittock N. Searle A. Croft M. Brewer C. Cole M. Eye. 2007; 21: 1220-1225Crossref PubMed Scopus (22) Google Scholar). We show here through extensive mutational and biochemical analysis that the linker regions in two receptor GCs, GC-C and guanylyl cyclase A (GC-A), play an important role in repressing the catalytic activity of the receptors in the absence of their ligands. In addition, our results provide for the first time a molecular explanation for detergent-enhanced guanylyl cyclase activity in this family of receptors and suggest a mechanism for this activation that could involve a hydrophobic interaction between the linker region and the guanylyl cyclase domain.DISCUSSIONThis is the first comprehensive study on the role of the putative coiled coil region in receptor GCs. Based on the phenotypes of the mutations that were observed and a lack of similarity in properties of mutants generated in equivalent positions in a coiled coil, we suggest that this region in guanylyl cyclases may not adopt a classical coiled coil helical structure. Instead, we suggest that a helix is formed by this linker region, where the amphipathic nature of the helix presents a hydrophobic stretch that may interact with regions on the guanylyl cyclase domain, thereby acting as a clamp to ensure low levels of cGMP production.High intracellular cGMP accumulation would result from conversion of MgGTP to cGMP, as mm concentrations of Mg2+, but only trace Mn2+, are found within the eukaryotic cell (41Finney L.A. O'Halloran T.V. Science. 2003; 300: 931-936Crossref PubMed Scopus (907) Google Scholar). The fact that all the mutant receptors possessed in vitro guanylyl cyclase activity when measured using MnGTP as a substrate showed that the cyclase domains were able to dimerize in a functional manner when Mn2+ was present as the metal co-factor. Perhaps the larger size and flexible co-ordination geometry of Mn2+ (42Seebeck B. Reulecke I. Kämper A. Rarey M. Proteins. 2008; 71: 1237-1254Crossref PubMed Scopus (52) Google Scholar) allows it to bind and form a functional catalytic site even in the presence of mutations that render the guanylyl cyclase domain poorly active when measured with MgGTP as a substrate. Nevertheless, because some linker mutant receptors showed robust guanylyl cyclase activity even when MgGTP alone was used as a substrate, we suggest that the linker region has an inhibitory role on the receptor guanylyl cyclase domain, perhaps by preventing the two catalytic domain subunits from juxtaposing themselves in a way suitable for catalysis in the absence of the ligand.Interestingly, interspersed among the high basal mutants were residues which when mutated to proline reduced the guanylyl cyclase activity to levels seen in the wild type receptor. Based on the periodicity that is observed, it is tempting to suggest that this region of the receptor (residues Tyr-771 to His-777) could show an alteration in the regular α-helical structure and generate interactions similar to those seen in a 310 helix. It is not clear, however, if this structural variation is seen in the basal conformation of the receptor or is a prerequisite structural transition mimicking the ligand-activated state of the receptor.We show that the phenotypes of mutations at equivalent positions in GC-C and GC-A are similar, indicating conservation of the role of these residues in these receptors. Naturally occurring mutations in the linker region of RetGC-1 have been identified in patients with autosomal cone-rod dystrophy (43Kitiratschky V.B. Wilke R. Renner A.B. Kellner U. Vadalà M. Birch D.G. Wissinger B. Zrenner E. Kohl S. Invest. Ophthalmol. Vis. Sci. 2008; 49: 5015-5023Crossref PubMed Scopus (55) Google Scholar, 44Koch K.W. Duda T. Sharma R.K. Mol. Cell. Biochem. 2002; 230: 97-106Crossref PubMed Scopus (63) Google Scholar). All mutations include a change in the Arg-838 residue, corresponding to the Arg-782 position in GC-C. These mutations showed guanylyl cyclase activity when measured using MnGTP in one study (45Duda T. Krishnan A. Venkataraman V. Lange C. Koch K.W. Sharma R.K. Biochemistry. 1999; 38: 13912-13919Crossref PubMed Scopus (39) Google Scholar) or MnGTP and the non-ionic detergent Triton X-100 in other studies (16Ramamurthy V. Tucker C. Wilkie S.E. Daggett V. Hunt D.M. Hurley J.B. J. Biol. Chem. 2001; 276: 26218-26229Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 46Tucker C.L. Woodcock S.C. Kelsell R.E. Ramamurthy V. Hunt D.M. Hurley J.B. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 9039-9044Crossref PubMed Scopus (88) Google Scholar). Because all the mutants in GC-C were active in the presence of MnGTP and we saw detergent-mediated effects when MgGTP was used as a substrate, we cannot comment on whether mutations in RetGC-1 abrogated detergent-stimulated activity as is seen with the R782P mutant in GC-C. However, mutant receptors showed ∼3-fold reduction in basal activity in comparison to the wild type receptor when assayed in the presence of MgGTP (45Duda T. Krishnan A. Venkataraman V. Lange C. Koch K.W. Sharma R.K. Biochemistry. 1999; 38: 13912-13919Crossref PubMed Scopus (39) Google Scholar). In none of these studies were intracellular levels of cGMP measured in cells overexpressing the mutant receptors, possibly because RetGC-1 is activated only in the presence of GCAPs. Mutations at Arg-838 showed an increased sensitivity to GCAP-1 stimulation of cyclase activity, and the inhibition potential of Ca2+ was decreased (16Ramamurthy V. Tucker C. Wilkie S.E. Daggett V. Hunt D.M. Hurley J.B. J. Biol. Chem. 2001; 276: 26218-26229Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In these studies it was assumed that the coiled coil region forms a parallel dimer, and mutation of the critical Arg-838 disrupts the dimerization interface leading to movement of the C-terminal parts of the two helices away from each other. Molecular dynamic simulations with the modeled coiled coil region suggested that mutation of Arg-838 led to the lengthening of the coiled coil region, thereby affecting the regulation of cyclase domain by GCAP-1 and Ca2+. The distinct mechanisms of regulation of GC-C (by an extracellular ligand) and RetGC-1 (by the intracellular GCAPs) may result in different phenotypes in linker region mutants. However, we predict that the lack of activation by detergent in mutations at certain positions in the linker region could be seen in all receptor GCs including RetGC-1 when assayed in the presence of MgGTP.A mutation in the catalytic domain of GC-A (E974A) has been reported that results in phenotypes very similar to many of the constitutively active mutants in GC-C (and GC-A) described in the current study (47Wedel B.J. Foster D.C. Miller D.E. Garbers D.L. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 459-462Crossref PubMed Scopus (25) Google Scholar). Full-length GC-AE974A is constitutively active and shows no further ANP-mediated activation and no regulation by ATP. In addition, a construct (HCAT) that contains a fragment of the C-terminal region of the KHD, the entire linker region, and the guanylyl cyclase domain also shows higher in vitro guanylyl cyclase activity than the wild type protein (47Wedel B.J. Foster D.C. Miller D.E. Garbers D.L. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 459-462Crossref PubMed Scopus (25) Google Scholar). It is, therefore, conceivable that the Glu-974 residue may interact with residues in the linker region, and disruption of this interaction either by mutation of the Glu residue or by mutation of interacting residues in the linker region (suggested by our study) could result in the same phenotype. However, it is important to note that the N-terminal residues of the catalytic domain (to which the linker sequences are attached) may lie on a face of the protein opposite to that of the Glu-974 residue (based on modeling analysis; data not shown). Therefore, an alternative explanation can be provided for the phenotype of the E974A mutant by stating that mutations in the linker region, which result in constitutive activity of receptor guanylyl cyclases, bring about a conformational change in the cyclase domain involving the conserved residue at position Glu-974 in GC-A. Clearly these hypotheses are likely to be verified when the structure of a receptor guanylyl cyclase is described or combination mutations are made in both the linker and the Glu-974 residue in GC-A.Mutations in the linker region of GC-C caused essentially three distinct phenotypes, as summarized in Fig. 7A. The fact that very few mutants possessed wild type-like activity in terms of ligand and detergent stimulability indicated that this region is critical for proper functioning of receptor GCs. A number of mutants lay in the upper left hand quadrant of the graph, and these represented mutant receptors that had lost all ligand stimulated activity but retained detergent-stimulated guanylyl cyclase activity. The most interesting mutant receptors (with the exception of R773P) were those in the lower left hand quadrant of the graph, which showed highly elevated basal guanylyl cyclase activity and no further ligand- or detergent-mediated activation. This suggested to us that detergents activate the wild type receptor by disrupting hydrophobic interactions that are held in place by residues whose mutation results in high basal activity. It is important to note, however, that mutations of some charged residues as well as hydrophobic residues also resulted in high basal activity, indicating that these charged residues may play a role in correct positioning of the regulatory hydrophobic region.The fact that ATP-mediated regulation is also lost in the mutant receptors which have high basal activity suggests that the linker region could also interact with the KHD. Because the cyclase domains need to form head to tail dimers, we suggest that the linker region could form anti-parallel helices, one face of which lies along the active site of the cyclase domain. The other face of the linker region could juxtapose to the KHD, with the KHD domains lying parallel to each other. We suggest this topology based on our mutational analysis and the effects that mutations in the linker region have on both ATP-mediated interaction with the KHD and guanylyl cyclase activity.An example of a nucleotide cyclase whose activity is regulated by sequences N-terminal to the cyclase domain is well demonstrated both biochemically and structurally by Rv1264, a pH-sensing adenylyl cyclase from Mycobacterium tuberculosis. This enzyme has maximum activity at acidic pH (6.0) and has an N-terminal regulatory domain and a C-terminal cyclase domain connected by a small linker (48Tews I. Findeisen F. Sinning I. Schultz A. Schultz J.E. Linder J.U. Science. 2005; 308: 1020-1023Crossref PubMed Scopus (101) Google Scholar). The N-terminal domain is autoinhibitory to the cyclase domain of the enzyme (49Linder J.U. Schultz A. Schultz J.E. J. Biol. Chem. 2002; 277: 15271-15276Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), and interestingly, a mutation in this region of Rv1264 (D107A) led to an increase in Vmax (3-fold) with little change in Km. The full-length crystal structure of Rv1264 revealed the conformational changes that are responsible for the pH-dependent switch between the active and inactive states of the enzyme. In the inactive state the linker between the N terminus and the catalytic domain attains a helical conformation, and the same region is a random coil in the active state of the enzyme. This structural change along with others facilitates the cyclase domains from two monomers to come in an appropriate head-to-tail orientation in the active state (48Tews I. Findeisen F. Sinning I. Schultz A. Schultz J.E. Linder J.U. Science. 2005; 308: 1020-1023Crossref PubMed Scopus (101) Google Scholar). Therefore, evidence for dramatic structural changes brought about by residues N terminus to a nucleotide cyclase domain is available, strengthening our suggestion that such conformational changes can be speculated to occur in the linker region of GC-C in conjunction with the N-terminal KHD and the C-terminal guanylyl cyclase domain to attain a ligand-mediated activated state.Some Class III nucleotide cyclases show regulation of their catalytic activity by associated HAMP (histidine kinases, adenylyl cyclases, methyl accepting chemotactic receptors, and phosphatases) domains. For example, point mutations in the HAMP domain of the mycobacterial adenylyl cyclase Rv3645 that were designed to remove hydrophobic surfaces led to a stimulation of Rv3645 adenylyl cyclase activity. In contrast, similar mutations in the HAMP domain of Rv1318c only marginally increased the activity of the associated cyclase domain, indicating specificity in the regulation brought about by the HAMP domains (50Linder J.U. Hammer A. Schultz J.E. Eur. J. Biochem. 2004; 271: 2446-2451Crossref PubMed Scopus (36) Google Scholar).To date, two guanylyl cyclase domain structures have been solved (5Rauch A. Leipelt M. Russwurm M. Steegborn C. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 15720-15725Crossref PubMed Scopus (87) Google Scholar, 6Winger J.A. Derbyshire E.R. Lamers M.H. Marletta M.A. Kuriyan J. BMC Struct. Biol. 2008; 8: 42Crossref PubMed Scopus (79) Google Scholar). The Cya2 (cyanobacterial) catalytic domain structure is similar to the structure of the activated adenylyl cyclase, whereas the Cyg12 (Synechocystis) catalytic domain is thought to be in the inactive conformation (because of dimethylarsenic additions to cysteine residues). We modeled the cyclase domain of GC-C into these two cyclase structures to generate inactive and active states to hypothesize on the role of the linker region in activating the cyclase domain. In the model of the active (Cya2) conformation of GC-C, helix α1 is found closer to the active site, and helix α4 is in the second monomer compared with the inactive (Cyg12) structure (Fig. 7B). This movement as suggested by Winger et al. (6Winger J.A. Derbyshire E.R. Lamers M.H. Marletta M.A. Kuriyan J. BMC Struct. Biol. 2008; 8: 42Crossref PubMed Scopus (79) Google Scholar) is important for positioning the nucleotide correctly in the active site, allowing catalysis. It was also suggested that regulatory proteins/domains can interact with the cyclase domain by docking onto the cavity formed between the α1-α2 loop and α3-β4 loop and regulating the activity of the cyclase domain, as seen with the adenylyl cyclase. It is possible that the linker region, which can form helical structures in the circular dichroism experiment, interacts with the cyclase domain, similar to the docking of the Gsα switch II helix in adenylyl cyclases (36Tesmer J.J. Sunahara R.K. Gilman A.G. Sprang S.R. Science. 1997; 278: 1907-1916Crossref PubMed Scopus (666) Google Scholar). However, given the limitations of computational methods to model loops in protein tertiary structure, it is not possible for us to conclusively say that this is the mode of regulation of the cyclase domain of receptor GCs by the associated linker region. Taken together, the mechanisms of activation of the cyclase domain remain speculations as of now and await the structural determination of a guanylyl cyclase domain with the regulator linker region and of course the entire intracellular domain. Nevertheless, we have shown in this study that the linker region of receptor GCs has a critical role to play in regulating not only the guanylyl cyclase activity of these receptors but also in the relay of conformational changes that occur from the extracellular domain and the KHD to the cyclase domain. In transmembrane receptors a series of conformational changes are required to transmit the information of ligand binding (an extracellular signal) to the interior of the cell, resulting in either altered interaction with signaling intermediates or in the regulation of a catalytic activity present in the receptor. In these multidomain receptors, where the ligand binding and effector domains are present in the same polypeptide chain, the relay of conformational changes is under the exquisite control of post-translational modifications or precise structural alterations. Receptor guanylyl cyclases (GCs) 4The abbreviations used are: GCguanylyl cyclaseGC-Aguanylyl cyclase AGC-Cguanylyl cyclase C (heat-stable enterotoxin receptor)GCAP-1GC-activating protein 1GSTglutathione S-transferaseANPatrial natriuretic peptideKHDkinase homology domainRetGC-1retinal guanylyl cyclaseSTheat-stable enterotoxinHAMPhistidine kinases, adenylyl cyclases, methyl accepting chemotactic receptors, and phosphatases. 4The abbreviations used are: GCguanylyl cyclaseGC-Aguanylyl cyclase AGC-Cguanylyl cyclase C (heat-stable enterotoxin receptor)GCAP-1GC-activating protein 1GSTglutathione S-transferaseANPatrial natriuretic peptideKHDkinase homology domainRetGC-1retinal guanylyl cyclaseSTheat-stable enterotoxinHAMPhistidine kinases, adenylyl cyclases, methyl accepting chemotactic receptors, and phosphatases. have an N-terminal extracellular ligand binding domain, a single transmembrane domain, and a C-terminal intracellular domain (1Padayatti P.S. Pattanaik P. Ma X. van den Akker F. Pharmacol. Ther. 2004; 104: 83-99Crossref PubMed Scopus (46) Google Scholar). Binding of ligands to the extracellular domain elicits a conformational change that increases the guanylyl cyclase activity of the receptor, resulting in increased cGMP production. The intracellular domain of receptor GCs contains a region that shares considerable sequence similarity to protein kinases and is referred to as the kinase homology domain (KHD). Binding of ATP to the KHD induces a conformational change that regulates cGMP production by the guanylyl cyclase domain (2Jaleel M. Saha S. Shenoy A.R. Visweswariah S.S. Biochemistry. 2006; 45: 1888-1898Crossref PubMed Sco