Key points are not available for this paper at this time.
The natriuretic peptide receptor-A (NPR-A) is composed of an extracellular domain with a ligand binding site, a transmembrane-spanning domain, a kinase homology domain, and a guanylyl cyclase domain. In response to agonists (atrial natriuretic peptide (ANP) and brain natriuretic peptide), the kinase homology domain-mediated guanylate cyclase repression is removed, which allows the production of cyclic GMP. Previous work from our laboratory strongly indicated that agonists are exerting their effects through the induction of a juxtamembrane dimeric contact. However, a direct demonstration of this mechanism remains to be provided. As a tool, we are now using the properties of a new mutation, D435C. It introduces a cysteine at a position in NPR-A corresponding to a supplementary cysteine found in NPR-C6, another receptor of this family (a disulfide-linked dimer). Although this D435C mutation only leads to trace levels of NPR-A disulfide-linked dimer at basal state, covalent dimerization can be induced by a treatment with rat ANP or with other agonists. The NPR-AD435C mutant has not been subjected to significant structural alterations, since it shares with the wild type receptor a similar dose-response pattern of cellular guanylyl cyclase activation. However, a persistent activation accompanies NPR-AD435C dimer formation after the removal of the inducer agonist. On the other hand, a construction where the intracellular domain of NPR-AD435C has been truncated (ΔKCD435C) displays a spontaneous and complete covalent dimerization. In addition, the elimination of the intracellular domain in wild type ΔKC and ΔKCD435C is associated with an increase of agonist binding affinity, this effect being more pronounced with the weak agonist pBNP. Also, a D435C secreted extracellular domain remains unlinked even after incubation with rat ANP. In summary, these results demonstrate, in a dynamic fashion, the agonistic induction of a dimeric contact in the juxtamembrane domain of NPR-A. In addition, this process seems to require membrane attachment of the receptor. Finally, the intracellular domain represses this contact at the basal state, showing its potent influence on the outer juxtamembrane domain. The natriuretic peptide receptor-A (NPR-A) is composed of an extracellular domain with a ligand binding site, a transmembrane-spanning domain, a kinase homology domain, and a guanylyl cyclase domain. In response to agonists (atrial natriuretic peptide (ANP) and brain natriuretic peptide), the kinase homology domain-mediated guanylate cyclase repression is removed, which allows the production of cyclic GMP. Previous work from our laboratory strongly indicated that agonists are exerting their effects through the induction of a juxtamembrane dimeric contact. However, a direct demonstration of this mechanism remains to be provided. As a tool, we are now using the properties of a new mutation, D435C. It introduces a cysteine at a position in NPR-A corresponding to a supplementary cysteine found in NPR-C6, another receptor of this family (a disulfide-linked dimer). Although this D435C mutation only leads to trace levels of NPR-A disulfide-linked dimer at basal state, covalent dimerization can be induced by a treatment with rat ANP or with other agonists. The NPR-AD435C mutant has not been subjected to significant structural alterations, since it shares with the wild type receptor a similar dose-response pattern of cellular guanylyl cyclase activation. However, a persistent activation accompanies NPR-AD435C dimer formation after the removal of the inducer agonist. On the other hand, a construction where the intracellular domain of NPR-AD435C has been truncated (ΔKCD435C) displays a spontaneous and complete covalent dimerization. In addition, the elimination of the intracellular domain in wild type ΔKC and ΔKCD435C is associated with an increase of agonist binding affinity, this effect being more pronounced with the weak agonist pBNP. Also, a D435C secreted extracellular domain remains unlinked even after incubation with rat ANP. In summary, these results demonstrate, in a dynamic fashion, the agonistic induction of a dimeric contact in the juxtamembrane domain of NPR-A. In addition, this process seems to require membrane attachment of the receptor. Finally, the intracellular domain represses this contact at the basal state, showing its potent influence on the outer juxtamembrane domain. natriuretic peptide receptor rat NPR atrial natriuretic peptide rat ANP (residues 1–28) or natriuretic peptide A porcine brain natriuretic peptide (residues 1–32) C-type natriuretic peptide (residues 1–22) atriopeptin I (rANP residues 5–25) des-Gln18,Ser19,Gly20,Leu21,Gly22ANP 4–23-NH2 (rat) polyacrylamide gel electrophoresis bovine serum albumin 1-methyl-3-isobutylxanthine extracellular domain transmembrane domain kinase homology domain guanylyl cyclase domain His-tagged ECD Dulbecco's modified Eagle's medium erythropoietin receptor The natriuretic peptide receptors (NPRs)1 are members of a family of single-transmembrane domain receptors that mediate their effects through the production of cyclic GMP (1Garbers D.L. Methods. 1999; 19: 477-484Crossref PubMed Scopus (54) Google Scholar). Three different NPRs have been identified, and two of these, NPR-A and NPR-B, respond to agonists by the activation of their guanylyl cyclase catalytic domain. The production of intracellular cGMP mediates their effects on diuresis, vasorelaxation, and the inhibition of the renin-angiotensin-aldosterone system (2Chinkers M. Annu. Rev. Biochem. 1991; 60: 553-575Crossref PubMed Scopus (201) Google Scholar). A third receptor, called NPR-C or the clearance receptor, displays only 37 amino acids in its intracellular domain and is devoid of guanylyl cyclase activity. NPR-C is a disulfide-bridged dimer that internalizes through a fast intracellular cycle process (3Cohen D. Young G.Koh Nikonova L.N. Gordon P. orter J. Maack T. J. Biol. Chem. 1996; 271: 9863-9869Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and might be involved in signal transduction (4Anand-Shrivastava M.B. Sehl P.D. Lowe D.G. J. Biol. Chem. 1996; 271: 19324-19329Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). NPR-A is stimulated by two peptides, ANP and BNP, whereas CNP is the only known agonist of NPR-B (2Chinkers M. Annu. Rev. Biochem. 1991; 60: 553-575Crossref PubMed Scopus (201) Google Scholar, 5Nakao K. Ogawa Y. Shin-ichi S. Imura H. J. Hypertens. 1992; 10: 907-912Crossref PubMed Scopus (209) Google Scholar). NPR-C has nearly equal binding affinity for all of these natriuretic peptides (6Itakura M. Iwashina M. Mizuno T. Teizo I. Hagiwara H. Hirose S. J. Biol. Chem. 1994; 269: 8314-8318Abstract Full Text PDF PubMed Google Scholar,7Suga S. Nakao K. Hosada K. Mukoyama M. Ogawa Y. Shirakami G. Arai H. Saito Y. Kambayashi Y. Inouye K. Imura H. Endocrinology. 1992; 130: 229-239Crossref PubMed Google Scholar). NPR-A is an ∼130-kDa protein that contains four structural domains: an extracellular domain (ECD) with a ligand binding site, a transmembrane domain (TM), a kinase homology domain (KHD), and a guanylyl cyclase domain (GC) (2Chinkers M. Annu. Rev. Biochem. 1991; 60: 553-575Crossref PubMed Scopus (201) Google Scholar). Several studies have demonstrated that this receptor is spontaneously preassociated in noncovalent dimers or oligomers (8Chinkers M. Wilson E.M. J. Biol. Chem. 1992; 267: 18589-18597Abstract Full Text PDF PubMed Google Scholar, 9Wilson E.M. Chinkers M. Biochemistry. 1995; 34: 4696-4701Crossref PubMed Scopus (153) Google Scholar, 10Thompson D.K. Garbers D.L. J. Biol. Chem. 1995; 270: 425-430Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Taken together, these studies have indicated that both extracellular and intracellular domains are involved in NPR-A dimerization. According to the current model of agonist activation, NPR-A signal transduction includes these five sequential steps (11Jewett J.R.S. Koller K.J. Goeddel D.V. Lowe D.G. EMBO J. 1993; 12: 769-777Crossref PubMed Scopus (71) Google Scholar). 1) The binding of the natriuretic peptide to the ectodomain induces a conformational change. 2) This modification corresponds to a signal that migrates through the TM domain. 3) The KHD responds to this signal by adopting a conformation that allows ATP binding. 4) ATP binding has two major effects in derepressing the guanylyl cyclase activity and increasing the off-rate of ANP from the receptor (12Larose L. McNicoll N. Ong H. De Léan A. Biochemistry. 1991; 30: 8991-8996Crossref Scopus (53) Google Scholar). 5) Subsequent desensitization results from reduction in phosphorylation state of the KHD (13Potter L.R. Garbers D.L. J. Biol. Chem. 1992; 267: 14531-14534Abstract Full Text PDF PubMed Google Scholar, 14Potter L.R. Hunter T. Methods. 1999; 19: 506-520Crossref PubMed Scopus (33) Google Scholar). We have previously brought out the remarkable conservation of spacing between the cysteine residues found in the extracellular domain of nearly all of the guanylyl cyclases (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). we a the two by which are in nearly all of the juxtamembrane domains of NPRs two have been to be through an in rat NPR-A M. PubMed Scopus Google Scholar). On the other hand, we out a found in the receptor, where the juxtamembrane cysteine is (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) the only juxtamembrane cysteine in is to an this receptor is found a covalent (6Itakura M. Iwashina M. Mizuno T. Teizo I. Hagiwara H. Hirose S. J. Biol. Chem. 1994; 269: 8314-8318Abstract Full Text PDF PubMed Google Scholar). to the cysteine of we previously the mutation in which its juxtamembrane cysteine (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The in the of this cysteine to in it of the cysteine in to of this mutation to a spontaneously disulfide-bridged This mutant found to be and it an increase in the binding affinity of a weak agonist (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). these we a model where agonists are a dimeric in the juxtamembrane of catalytic activation of the guanylyl However, we indicated at the that we not the of a conformational induced by the mutation of the (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). it not known the of the might by in the activation of In the current our is to that a juxtamembrane dimerization is associated with NPR-A activation. structural alterations, we to the of the we to a of NPR-C that displays a supplementary juxtamembrane cysteine an T. Iwashina M. M. Hagiwara H. Hirose S. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). the juxtamembrane of and this supplementary cysteine in with the in NPR-A We that the of a cysteine at position might to a NPR-A. We a disulfide-linked We found that this mutant displays only trace levels of spontaneous covalent agonists can a dimeric This to involved in receptor activation. in the The construction of the His-tagged has been (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). This includes all of the extracellular domain to by by the in with the using the from NPR-AD435C by in using the A mutant of the intracellular domain of NPR-A by The using a and two The and the other in The by the a and a The This to in which been previously with the The construction the the transmembrane-spanning domain, and the two intracellular residues by the and a The by the using NPR-AD435C an The and the by on the two using the from The in Dulbecco's modified Eagle's medium with bovine serum and of in a at 37 the cyclic GMP of the NPR-A and NPR-AD435C at the of and by the of in using the D. T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). and of the truncated receptors the of to be to a of with the receptor. This by of or with of the the of the and the with of and in in wild type NPR-A and NPR-AD435C in and to to the with of the medium and to of to The induction to for in a at 37 the with and all The in a at membrane the on and of The in for with a and for at The in and the and at The protein using the protein The induction of receptor dimerization by after the of membrane on a in the or of in the for the binding studies and the in induction of for ΔKC and or at for the NPR-A and the with and in and the The in for with a and for at The and in the Finally, in and the in and at and from medium of the in and at The His-tagged on gel (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). from the gel with The in using of of membrane from NPR-A or NPR-AD435C to of incubation and the agonist. the atriopeptin I or in the incubation The agonist induction of to for at the in a at The in and The for The covalent dimerization by after the of membrane on a The induction of at or at with for in of binding The of covalent dimer by after the of on a using the (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). to at for in of binding and by incubation of of membrane or ΔKCD435C with of and increasing of from ligand by on with and to to on The with and in a of of the medium and to of of the medium and extracellular cyclic GMP by J. S. Ong H. A. Google Scholar). the to The wild type and mutant receptor levels by which to their cGMP NPR-A and NPR-AD435C to to on the with of and to the The incubation to for in a at 37 The with and for another this the with that and in and the The protein and these for guanylyl cyclase in other studies (13Potter L.R. Garbers D.L. J. Biol. Chem. 1992; 267: 14531-14534Abstract Full Text PDF PubMed Google Scholar, L.R. Hunter T. Biol. PubMed Scopus Google Scholar). of membrane at 37 in with of and using or by and ATP or with of GMP from by on and by previously J. S. Ong H. A. Google Scholar). on in the or of in the The to a membrane using the of NPR-A and ΔKC using a the and by affinity This corresponds to NPR-A by a for The rat NPR-A from this at a both receptors are signal with an to the the His-tagged and on a in the In this in the and in the since it found to the to a membrane and the using a to the by the signal with a using the with the for on the Léan A. Google Scholar). binding with the on a model for the of Léan A. D. J. PubMed Google Scholar). the binding dose-response and are in the We the that the of a cysteine at position a covalent dimerization of This of its with an cysteine involved in the of dimers studies with in only levels of spontaneous dimerization. this we to and the that dimeric might be induced by the agonist of NPR-A. This out to be since dimers of be after a cellular incubation with 37 We this induction through a using a NPR-AD435C A signal of corresponding to NPR-AD435C dimers is on It is that an induction is not in wild type NPR-A. A of the dimeric induction an of which is the in cellular guanylyl cyclase The for this is not However, the between to formation be a of the process and might the this supplementary an an results are showing that ANP induces a dimeric contact in the juxtamembrane domain of This is in with our on the of mutant (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the dimerization properties of NPR-AD435C this the of NPR-AD435C covalent dimerization at basal state the of a that represses this contact in of ANP. the D435C mutation the NPR-A we cellular guanylyl cyclase activation by As in the for the wild type and the mutant are and their levels of are It be that we a increase in the basal activity of NPR-AD435C with that of This increase might that the D435C mutation has modified the in the juxtamembrane it be to a trace of NPR-AD435C dimer on through signal this it can be that NPR-AD435C displays a response to that is to that for wild type NPR-A. the on the results with this mutant are to be to the wild type receptor. We the induction of NPR-AD435C covalent dimerization with persistent activation. cyclase activity in membrane from and NPR-AD435C for with ANP. of the ligand through of by ligand and membrane The to using or with ATP and The results a of the activation with This NPR-A to its catalytic and is an of activity. Several can be from the the of with ATP ANP to in from This be to desensitization of the receptor. Also, in cellular ANP treatment results in an increase of basal activity that is for NPR-AD435C for the wild Finally, ATP is for both receptors a of cellular ANP This has been previously receptor desensitization of NPR-A (13Potter L.R. Garbers D.L. J. Biol. Chem. 1992; 267: 14531-14534Abstract Full Text PDF PubMed Google Scholar). However, this increase in ATP response is in the D435C mutant in the wild type receptor results a more pronounced of NPR-AD435C persistent activation a of ANP We the dimerization in in membrane A induction of a dimer can be are with for at induction is not in the wild type Although dimerization is not complete at the ANP an of can be This is from is in cellular activation However, at the dimerization is more complete is in has been for the cellular dimeric it is that the of incubation to protein might or the of formation of the which be in these to the of the we the induction using the of the natriuretic peptide receptors and A of of the different peptides to the of the dimerization in these this to the of the agonists. As in the of induction with agonists on NPR-A (rANP CNP and this we to the of the intracellular domain in the of the NPR-AD435C covalent dimerization at the basal We with of the intracellular domain on wild type NPR-A and on NPR-AD435C for their of covalent dimerization. ΔKCD435C covalent dimerization complete on whereas remains the intracellular domain is at the of a that represses at basal On the other hand, the dimeric state, that a between the of the extracellular and the intracellular domains the juxtamembrane dimeric We to the effects of this from the intracellular domain on the of the extracellular domain. As a tool, we the binding properties of and which are and weak agonists of As we have the binding of is and can be and affinity (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M. L. M. De Léan A. Biochem. 1999; PubMed Scopus Google Scholar). The associated with this binding are not However, since the of affinity is in we have previously associated the affinity state with a juxtamembrane conformation corresponding to the state (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). We that the binding of more by the intracellular domain that of studies on and As in the binding of and on and NPR-AD435C are Also, the binding of on NPR-A and be with similar we the from of The corresponding to binding on and are However, the binding on the truncated receptors are showing pronounced to the and are nearly The corresponding for and ΔKCD435C are and On the other hand, the of binding on the two ΔKC are similar and only to the with receptors of these results that binding is more by the of the intracellular domain. It is that the on and ΔKCD435C are the binding on these truncated receptors is This that the in ΔKCD435C is not with the affinity conformation spontaneously by the receptor in the of the intracellular domain. In summary, these results that the ΔKC spontaneously a affinity It is to that the of the intracellular domain the receptor from spontaneously this state in the of agonist. We the D435C mutation to the dimerization of a secreted NPR-A ECD devoid of transmembrane domain. A His-tagged extracellular domain of NPR-AD435C in on and its of dimerization on As in only a trace of spontaneous dimer can be We for induction of dimerization with which to be induction at with the the cysteine in not a significant to an However, we this to a of noncovalent dimerization. we on gel is noncovalent dimerization and found that the dimerization complete after an incubation with results in response to ligand the juxtamembrane dimeric are different in the ECD receptor mutant with its membrane On the other hand, it is that the transmembrane domain of the receptor might influence the of the outer juxtamembrane its might be for induced of In this we have that agonists are a dimeric contact in the juxtamembrane domain of results that the intracellular domain the juxtamembrane associated with receptor activation. basal state, this the ectodomain agonist the is juxtamembrane dimerization receptor activation In that the induction of the dimeric in the NPR-AD435C a direct of these Finally, membrane of this mutant has to be for the covalent dimeric The spontaneous dimerization of ΔKCD435C that the ΔKC with transmembrane by a spontaneous juxtamembrane dimerization. On the other hand, our results a increase of binding affinity for both ΔKCD435C and with the receptor, whereas the affinity of ANP is not Taken together, these strongly that the ΔKC spontaneously a dimeric state, to the ectodomain dimeric that in the NPR-A. the of the intracellular domain more can of the of a of juxtamembrane dimeric which the intracellular might be by the influence of the ANP be more to this the affinity of an agonist might from a of its affinity for the ectodomain with its to the intracellular According to this the binding results with the are the of and for the the of affinity between and is for the ΔKC with the receptor which of the domain of NPR-A is involved in this can a potent influence of the it has been that the removal of the KHD leads to a activation of the K.J. De Lowe D.V. Biol. 1992; 12: PubMed Scopus Google M. Garbers D.L. PubMed Scopus Google Scholar). since the KHD seems to a potent on it might influence the ectodomain induction of in a receptor has been in other A. A. J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). In this have that in a mutant of the receptor a supplementary cysteine in the juxtamembrane domain. The induced dimer found to persistent kinase activity and affinity binding. The that the is a dimeric of the two corresponding to the a by has that the intracellular domain of the receptor its dimerization K.J. J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). found that the is from the receptor, the activation for a of Biochemistry. PubMed Scopus Google Scholar). that this might be to between of the which significant of the dimeric state of the A similar might the persistent activation that we have in the of with the wild type NPR-A this persistent activation in the of it is that the formation of the might of the dimeric studies have been on of the erythropoietin receptor activation D.L. Wilson 1999; PubMed Scopus Google Scholar, H. J. J. S. K. J. PubMed Scopus Google Scholar, D.L. Y. S. Wilson Biol. PubMed Scopus Google Scholar). a between agonist and juxtamembrane from the of with erythropoietin or with potent agonists. The have a model where the juxtamembrane domains of two are brought a in response to agonist induction Biol. 1999; PubMed Scopus (63) Google Scholar). This mechanism has been by in studies using a protein I. Wilson 1999; PubMed Scopus Google Scholar). It be that these studies are showing the intracellular domain to the agonistic of the In addition, of the intracellular domain of the is not to spontaneous juxtamembrane dimerization I. Wilson 1999; PubMed Scopus Google Scholar). the of in the seems to that in NPR-A. we are showing the intracellular domain of NPR-A a potent on its of the of the current NPR-A mutant is that ligand induction of covalent dimerization the of the between the in a is A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, N. D.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of by more of the protein N. D.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of formation might be by the of of the and the of structural that between the residues N. D.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). This might have the of dimer formation in our in at It is that studies with the receptor have that a reduction of the of receptor which might be to structural to the state K.J. J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the cellular induction of NPR-AD435C dimerization is after a incubation at 37 a of these In our we the of NPR-A which the other cysteine to an This dimer an activity (15Labrecque J. McNicoll N. Marquis M. De Léan A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). We that the activation are a dimeric of the juxtamembrane domain of NPR-A. However, we that we not the that the mutation induced a conformational that the receptor of the our T. Biochemistry. 1999; PubMed Scopus Google Scholar) the mutant which both juxtamembrane this mutant indicated that the in not for the activation. the mutant not in their which have this and to their that the of the the of the receptor in this which is for receptor In of the results that we are it is that this structural modification in the increase of the spontaneous of the M. PubMed Scopus Google Scholar) have the of the NPR-A extracellular domain. This is to and a spontaneous noncovalent dimerization of NPR-A ECD has been to at protein of N. N. Biochemistry. 1999; PubMed Scopus Google Scholar). The (residues a which from our complete the dimeric the the of of the from the known of the this a of between the of the two at position and that their to an results these structural As in response to agonist the of of the receptor a of On the other hand, the by the structural is not to mediate the at this with the of covalent dimerization in the that the conformation of the ECD not to the state of the receptor. to our it is more that the ΔKC spontaneously the This a significant influence of the transmembrane membrane on the and of the juxtamembrane In this with NPR-AD435C to that in this receptor. Also, it has with the to an activation model by the of a dimeric this of a significant to the of NPR-A. In summary, these results that agonists a dimeric in the juxtamembrane domain of an that is to its activation
Labrecque et al. (Thu,) studied this question.