- Email: [email protected]
Role of the amino terminus in ligand binding for the angiotensin II type 2 receptor
Molecular Brain Research 57 Ž1998. 325–329
Short communication
Role of the amino terminus in ligand binding for the angiotensin II type 2 receptor Daniel K. Yee, Jennifer N. Heerding, Marc Z. Krichavsky, Steven J. Fluharty
)
Departments of Animal Biology, Pharmacology, Psychology, and the Institute of Neurological Sciences, UniÕersity of PennsylÕania, Philadelphia, PA 19104, USA Accepted 7 April 1998
Abstract Key amino terminal residues in type 1 ŽAT1 . angiotensin II ŽAngII. receptors are not conserved within type 2 ŽAT2 . receptors. We therefore characterized amino terminal mutants that are transiently expressed in COS-3 membranes. AT2 amino terminal deletion drastically reduced affinity for AngII, suggesting its importance for this subtype. AT1 –AT2 amino terminal exchanges retained wild type AngII affinities Ž K d ranging from 3–5 nM., indicating compensation despite substantial sequence dissimilarities. Finally, binding of AT2 selective ligands ŽCGP42112A and PD123319. was not dependent on the amino terminus. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Angiotensin; Amino terminus; Mutagenesis; Ligand binding
Angiotensin II ŽAngII. is one of the key hormones involved in the regulation of cardiovascular and body fluid homeostasis. This peptide hormone affects numerous peripheral target organs, as well as discrete areas of the brain, through its interactions with cell surface receptors at these locations w15x. At least two main families of AngII receptors, referred to as type 1 ŽAT1 . and type 2 ŽAT2 ., have been identified through the use of subtype selective ligands w3x. With the cloning of these AngII receptor subtypes, more detailed structural information on these receptors has become available w11–13,16x. Both subtypes conform to the seven transmembrane-spanning structural motif of G-protein coupled receptors. Mutagenesis of G-protein coupled receptors has yielded valuable insights into the structural and molecular properties of this superfamily of receptors. With respect to AngII receptors, these studies have focused primarily on the AT1 receptor subtype and have identified several structural components that are involved in ligand binding and receptor function w2,8,10,14,17–20x. In contrast to the accumulating data on the structural and molecular properties of the
) Corresponding author. Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104. Fax: q1-215-898-0899; E-mail: [email protected]
0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 1 0 4 - 1
AT1 receptor, the AT2 receptor has been largely neglected with respect to structural analysis by either mutagenesis or molecular modeling. Both the AT1 and AT2 receptors bind AngII with nearly identical affinities, despite sharing only a 34% homology. Given the relatively low level of homology between the AT1 and AT2 receptors, it seems plausible that areas of highest homology may confer AngII binding specificity to the two receptor subtypes. Indeed, a comparison of the amino acid sequences show that some of the key residues identified by AT1 mutagenesis experiments as critical for AngII binding are shared by the two receptor subtypes w8,20x. However, there are other identified AT1 residues that are not conserved in the AT2 subtype. One such area is the amino terminus, which is highly divergent in the two receptor subtypes. Moreover, the AT2 amino terminus is almost twice the size of the analogous AT1 region Žsee Fig. 1.. The importance of this protein domain for high affinity AngII binding in the AT1 subtype has been clearly established by analysis of a series of point mutations in the AT1 amino terminus w8x. However, these critical AT1 residues identified by mutagenesis studies are not conserved in the AT2 receptor Žsee Fig. 1. and therefore high affinity binding to AngII in this receptor family cannot be solely explained by the actions of a few conserved residues in the two subtypes. Since there is currently little information on
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D.K. Yee et al.r Molecular Brain Research 57 (1998) 325–329
Fig. 1. Alignment of amino acid sequences of AT1 and AT2 amino terminal domains. The amino acid sequences of mouse AT1A and AT2 receptors are compared over their amino terminal regions. Skipped sequences as indicated by hyphens are introduced for maximizing sequence homology. Vertical lines Ž<. between the sequences highlight identical residues between the two subtypes. Previous mutagenesis experiments of the AT1 receptor by Hjorth ¨ et al. w8x have identified several residues that appear to be involved in the binding of AngII. These key AT1 residues are indicated above with asterisks Ž)..
the binding epitopes for the AT2 receptor, any possible contribution towards ligand binding by its amino terminus remains to be established. Consequently, in this paper, we have constructed and characterized an amino terminal deletion of the AT2 receptor subtype as well as generated and analyzed the binding properties of chimeric exchanges of this protein domain between the two receptor subtypes Žsee Fig. 2.. The polymerase chain reaction ŽPCR. was used to create all mutational constructs with AT1 and AT2 receptor cDNAs that have previously been isolated from the murine neuroblastoma N1E-115 cell line w6,21x serving as templates. To construct the AT2 amino terminal deletion delŽ5–40.AT2 , a specially designed forward primer 5XGCGGGATCCAAAATGAAAGATAATTTGGAAGCAATTCCTGTTCTCTAC-3X was employed that preserved the first four amino acids, while removing the following 36 amino acids of the AT2 amino terminus ŽMet-Lys-AspAsn-rest of AT2 receptor.. A complete removal of the amino terminus was not employed because of the concern that such an extreme deletion may interfere with translocation of the mutant receptors to the plasma membrane. Thus, the AT2 deletion mutant Ždel 5–40.AT2 retains the first four amino acids of the amino terminus with the expectation that this construct would not be as disruptive to normal cellular processing as removal of the entire protein domain Žsee Fig. 2.. The amino terminal exchange mutants were generated by the splicing by overlap extension ŽSOE. process w9x. In attaching the AT2 amino terminus onto the AT1 receptor, the overlapping primers 5X-CATGACAAAATGCTTATCTGATGGTTTGTG-3X and 5X-TCAGATAAGCATTTTGTCATGATCCCTACTCTC-3X were used in the SOE reaction. In constructing the AT1 amino terminus onto an AT2 receptor the overlapping primers 5X-TGCTTCCAATATGTAACTGTGCCTGCCAGC-3X and 5X-CACAGTTACATATTGGAAGCAATTCCTGTTCTC-3X were used. A small amount of Pfu, a thermostable, new generation DNA polymerase was also added to the polymerase chain reactions Ž1:100 Pfu:Taq. in order to improve the fidelity of the SOE process w1,4x.
DNA sequencing of all mutant receptors was performed to verify that the desired mutations was produced without any inadvertant misincorporations due to PCR. Following verification, the mutant receptors were then directly ligated into the eukaryotic expression vector, PCR3 ŽInvitrogen.. Transfection of COS-3 cells were performed as previously described w22x. Subsequently, cell membranes were prepared from transfected cells and radioligand binding analysis was performed using monoiodinated 125 I-AngII ŽNENrDupont. as previously described w5,22x. Binding data was then analyzed by the program InPlot ŽGraphPad Software.. The necessity of the AT1 amino terminus for high affinity AngII binding has been previously established by conservative segment exchange and point mutations for this subtype w8x. In contrast, the role of the AT2 amino terminus, which is greatly divergent compared to the AT1 amino terminus, remains to be established. Consequently, in order to investigate a possible role of this protein domain in the AT2 subtype, the deletion mutant Ždel 5–40.AT2 was constructed that removed a large portion of the AT2 amino terminus. This mutant specifically bound 125 I-AngII, indicating that despite deletions of a large portion of the amino terminus, the mutant receptor protein was expressed and properly oriented in the plasma membrane. However, in marked contrast to the wild type receptor Ž K d s 3.78 " 0.15 nM; mean " S.E.M.; n s 3., 125 I-AngII binding was not saturable in the deletion mutant
Fig. 2. Creation of AT2 amino terminal deletion mutant and AT1 –AT2 amino terminal exchange mutants. To address the possible role of the amino terminus in ligand binding for AngII receptors, a deletion mutant for the AT2 amino terminus, as well as chimeric exchange of these protein domains between the AT1 and AT2 subtypes were constructed using the splicing by overlap method. These mutants are depicted above.
D.K. Yee et al.r Molecular Brain Research 57 (1998) 325–329
over the range of ligand concentration that was used Ždata not shown, n s 3.. Although the nonsaturable nature of the mutant binding does not permit a precise determination of receptor expression and ligand affinity by curve-fitting, this nonetheless indicates a drastic reduction in the affinity of the mutant receptor for the ligand Ž K d ) 100 nM, the limits of the curve fitting software over the concentration range of the ligands used.. Therefore, these data suggest that portions of the AT2 amino terminus are required to maintain high affinity binding of AngII. However, since the critical AT1 amino terminal residues are not conserved in the AT2 subtype, the AT2 amino terminus must interact with its ligand via an alternate binding mechanismŽs., i.e., through a different set of amino acid residues. To further investigate the role of the amino termini in the AngII receptor family, we constructed chimeric exchanges of this protein region between the two subtypes Žsee Fig. 2.. Saturation isotherms using 125 I-AngII were subsequently performed on the harvested membrane preparations from transfected COS-3 cells. Although receptor expression varied with transfection efficiency for each of the constructs, the binding for 125 I-AngII was saturable for each of the chimeric receptor and was thus amenable to analysis by the curve fitting software InPlot Žfrom GraphPad.. Bmax values ranged from 1333 to 1677 fmolrmg protein for the AT2 receptor, from 135.1 to 5482 fmolrmg protein for the AT1 receptor, from 37.7 to 261.5 fmolrmg protein for the chimeric AT2 receptor with a substituted AT1 amino terminus ŽAT1r2 NT., and from 8.94 to 29.7 fmolrmg protein for the chimeric AT1 receptor with a substituted AT2 amino terminus ŽAT2r1 NT.. The 125 IAngII binding affinities for the chimeric receptors were essentially identical to the wild type receptors Žunpaired t-test, p - 0.05.: wild type AT2 had a K d of 3.78 " 0.15 nM Ž n s 3. while that of AT1r2 NT was 5.06 " 2.49 Ž n s 3.; wild type AT1 had a K d of 3.23 " 1.78 nM Žmean " S.E.M.; n s 3. while that of AT2r1 NT was 3.26 " 1.17 nM Ž n s 3.. Since the importance of the amino termini in the binding of AngII is established for both subtypes and the structural dissimilarities between the two amino termini are so prominent, it is particularly striking that the chimeric exchanges of this protein domain among the two receptor subtypes retain wild type binding for AngII. These results suggest that although the two receptor subtypes may employ differing amino acid residues in the amino terminus to interact with AngII, these alternate binding mechanismŽs. can compensate for each other, most likely by interacting with a similar portion of the AngII molecule. It is important to note, however, that the loss-of-function data in previous AT1 mutagenesis experiments may simply have reflected global changes in receptor folding that perturbed AngII binding rather than the actual removal of specific binding epitopes in the AT1 amino terminus w8x Žalso see Fig. 1.. Although scanning alanine mutations successfully mapped the location of key AT1 residues in the amino
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terminus through the loss of peptidic binding and is therefore suggestive of the importance of these residues in ligand binding, this strategy cannot specify the nature of the interactions between these targeted residues and the ligand. Consequently, any conclusions drawn from these AT1 experiments must be cautiously considered and an alternate explanation for our chimeric receptor data is that despite the great dissimilarities in their sequences, the exchange of the native amino terminal regions of the AT1 and AT2 receptors could simply be less globally disruptive than the other mutations in previous AT1 experiments. Still, the retention of wild type binding by the chimeric receptors indicate that the amino terminus of one receptor subtype is not incompatible with the remaining protein molecule of the reciprocal subtype and therefore makes it likely that despite the low relative homology between the two subtypes, AngII is situated in the binding pocket of both subtype receptors in a similar general orientation. In their transmembrane domains and in other extracellular domains, the AT1 and AT2 receptors do share some conserved residues. Our previous mutational work on these conserved residues in the AT2 receptor have indicated that some, but not all of these conserved residues may play a role in the binding of AngII w7,22x. These earlier results suggested that despite the relatively low homology, the two AngII receptor subtypes may share some essential commonalities in the manner that they bind AngII and its analogs. Because several of the amino acid residues previously implicated in the binding of AngII by AT1 mutagenesis studies w8,20x are not conserved in the AT2 receptor, the AngII binding mechanisms for both subtypes cannot be entirely identical. As demonstrated by the amino terminal chimeric exchange experiments, other alternate mechanisms that are distinct to each subtype are also necessary and may have developed from the ongoing divergence of these two subtypes during evolution to maintain high affinity for AngII. In addition to investigating the binding of these receptor subtypes to an identical ligand, namely AngII, the amino terminal chimeric exchanges offered an opportunity to map the binding epitopes of subtype specific compounds. With respect to the AT1 receptor, the binding epitopes of its specific antagonist losartan have been extensively investigated and have been determined to reside exclusively within the transmembrane regions of the receptor w10,17,18x. In contrast, a similar understanding of binding of AT2 selective compounds for their subtype remains to be determined. In this regard, the chimeric exchange of the AT1 amino terminus onto an AT2 receptor ŽAT1r2 NT. provided an opportunity to investigate any possible contributions by this protein domain towards the binding of AT2 selective compounds. Therefore, competition binding analysis was performed comparing the AT1r2 NT mutant to the wild type AT2 receptor with respect to both AT1 and AT2 specific ligands. As shown in Fig. 3, replacement of the AT2 amino terminus with that of the AT1 did not
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Fig. 3. Competition of 125 I-AngII binding with various ligands in wild type AT2 and in chimeric AT2 receptor with a substituted AT1 amino terminus. Competition of 0.6 nM 125 I-AngII to wild type AT2 and to chimeric AT1r 2 amino terminal exchange mutant was determined in the presence of increasing concentrations from 10y1 1 to 10y6 M of AngII, losartan, CGP42112A, and PD123319. The competition curves shown above are representative of three independent experiments.
confer losartan binding to this chimeric receptor, confirming that the binding epitopes for this AT1 antagonist are not in the amino terminus. However, 125 I-AngII continued to be effectively displaced by AngII and the AT2 specific compounds CGP42112A and PD123319 for the AT1r2 NT mutant. The K i values for wild type AT2 receptor with respect to AngII, CGP42112A, and PD123319 were respectively 11.0 " 1.68 nM, 3.58 " 0.74 nM, and 19.7 " 2.97 nM Ž n s 4.; while for the chimeric receptor, they were respectively 2.0 " 0.20 nM, 0.695 " 0.083 nM, and 8.99 " 0.60 nM Ž n s 3.. Because small, non-peptide ligands tend to be seated more deeply than larger, peptidic compounds in the ligand binding pocket where residues of the transmembrane domains are primarily responsible for determining binding affinity and specificity, high affinity binding of the non-peptide compound PD123319 by the chimeric receptor was not surprising. On the other hand, high affinity binding of the larger, peptidic CGP42112A
was also preserved in the chimeric receptor, indicating that the binding epitopes that determine AT2 specificity of this ligand do not reside in the amino terminus. Initially, one would have expected that the peptidic CGP42112A would interact with the extracellular domains, including the amino terminus. It is possible that the amino terminus may still contribute towards the binding of CGP42112A, but because it is an analog of AngII this protein region may interact with a portion of CGP42112A that does not confer its subtype specificity. Thus, the chimeric mutant of an AT1 amino terminus onto an AT2 receptor may continue to displace 125 I-AngII with either AngII or CGP42112A with high efficiency. Although the rank order potency for AngII, CGP42112A, and PD123319 was identical for both receptors, these competitors did demonstrate greater efficiency in displacing 125 I-AngII in the chimeric receptor compared to the wild type receptor Žunpaired t-test, p - 0.05.. It is possible that the exchange of the long AT2 amino terminus with the shorter AT1 portion allowed for greater accessibility to the binding pocket for each of these ligands. The use of molecular biological techniques to introduce specific mutations into proteins has proven to be an invaluable research strategy in the analysis of protein structure and function. With respect to the AT1 receptor, these techniques have yielded a growing data set which have been used to define the ligand binding pocket for this receptor subtype. The experiments presented in this paper represent our continuing attempts to extend these approaches to the AT2 receptor subtype. These experiments on the amino terminal regions, a protein domain that greatly differs in the two subtypes, demonstrate that identification of a function, in this case, the high affinity binding of AngII, cannot simply be an inevitable consequence of sequence homology. In fact, the preservation of high affinity binding of AngII by the chimeric receptors indicate that the amino termini of AT1 and AT2 are able to functionally compensate for one another despite great structural and sequence dissimilarities. Yet both subtypes do have some residues in common in other parts of these receptor proteins, which may represent some structural commonalities in the AngII receptor family. Indeed, our initial mutagenesis studies of extracellular and transmembrane domains of the AT2 receptor have suggested that the two AngII receptor subtypes may share some structural commonalities in their binding mechanismŽs. for AngII w7,22x. Thus, evaluating structural similarities and differences between the two receptor subtypes should provide further insights towards understanding the molecular mechanisms that define ligand binding for the entire AngII receptor family.
Acknowledgements This work was supported by NS23986, MH43787, and by Grants-In-Aid from the American Heart Association ŽNational and Southeastern Pennsylvania Affiliate..
D.K. Yee et al.r Molecular Brain Research 57 (1998) 325–329
References w1x W.M. Barnes, PCR amplification of up to 35 kb DNA with high fidelity and high yield from lambda-bacteriophage templates, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 2216–2220. w2x C. Bihoreau, C. Monnot, E. Davies, B. Teutsch, K.E. Bernstein, P. Corvol, E. Clauser, Mutation of Asp74 of the rat angiotensin II receptor confers changes in antagonist affinities and abolishes Gprotein coupling, Proc. Natl. Acad. Sci. U.S.A. 90 Ž1993. 5133– 5137. w3x F.M. Bumpus, K.J. Catt, A.T. Chiu, M. DeGasparo, T. Goodfriend, A. Husain, M.J. Peach, D.G. Taylor, P.B. Timmermans, Nomenclature for angiotensin receptors. A report of the Nomenclature Committee of the Council for High Blood Pressure Research, Hypertension 17 Ž1991. 720–721. w4x S. Cheng, C. Fockler, W.M. Barnes, R. Higuichi, Effective amplification of long targets from cloned inserts and human genomic DNA, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 5695–5699. w5x S.J. Fluharty, L.P. Reagan, Characterization of binding sites for the angiotensin II antagonist 125 I-wSarc 1 ,Ile 8 x-angiotensin II on murine neuroblastoma N1E-115 cells, J. Neurochem. 52 Ž1989. 1393–1400. w6x P.F. He, X.D. Yang, Y.F. Guo, L.P. Reagan, D.K. Yee, S.J. Fluharty, Molecular cloning and expression of an angiotensin II type 1 receptor from murine neuroblastoma N1E-115 cells, Soc. Neurosci. Abstr. 20 Ž1994. 220.10. w7x J.N. Heerding, D.K. Yee, S.L. Jacobs, S.J. Fluharty, Mutational analysis of the angiotensin II type 2 receptor: contribution of extracellular amino acids, Regul. Pept. 72 Ž1997. 97–103. w8x S.A. Hjorth, ¨ H.T. Schambye, W.J. Greenlee, T.W. Schwartz, Peptide binding residues in the extracellular domain of the AT1 receptor, J. Biol. Chem. 269 Ž1994. 30953–30959. w9x R.M. Horton, H.D. Hunt, S.N. Ho, J.K. Pullen, L.R. Pease, Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension, Gene 77 Ž1989. 61–68. w10x H. Ji, M. Leung, Y. Zhang, K.J. Catt, K. Sandberg, Differential structural requirements for specific binding of nonpeptide and peptide antagonists to the AT1 angiotensin receptor—Identification of amino acid residues that determine binding of the antihypertensive drug Losartan, J. Biol. Chem. 269 Ž1994. 16533–16536. w11x Y. Kambayashi, S. Bardhan, K. Takahashi, S. Tsuzuki, H. Inui, T. Hamakubo, T. Inagami, Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine phosphatase inhibition, J. Biol. Chem. 268 Ž1993. 24543–24546.
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w12x M. Mukoyama, M. Nakajima, M. Horiuchi, H. Sasamura, R.E. Pratt, V.J. Dzau, Expression cloning of type 2 angiotensin II receptor reveals a unique class of seven-transmembrane receptors, J. Biol. Chem. 268 Ž1993. 24539–24542. w13x T.J. Murphy, R.W. Alexander, K.K. Griendling, M.S. Runge, K.E. Bernstein, Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor, Nature 351 Ž1991. 233–236. w14x K. Ohyama, Y. Yamano, S. Chaki, T. Kondo, T. Inagami, Domains for G-protein coupling in angiotensin II receptor type I: studies by site-directed mutagenesis, Biochem. Biophys. Res. Commun. 189 Ž1992. 677–683. w15x M.J. Peach, Renin–angiotensin system: biochemistry and mechanism of action, Physiol. Rev. 57 Ž1977. 313–370. w16x K. Sasaki, Y. Yamano, S. Bardhan, N. Iwai, J.J. Murray, M. Hasegawa, Y. Matsuda, T. Inagami, Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor, Nature 351 Ž1991. 230–233. w17x H.T. Schambye, S.A. Hjorth, D.J. Bergsma, G. Sathe, T.W. ¨ Schwartz, Differentiation between binding sites for angiotensin II and nonpeptide antagonists on the angiotensin II type 1 receptors, Proc. Natl. Acad. Sci. U.S.A. 91 Ž1994. 7046–7050. w18x H.T. Schambye, B.V. Wijk, S.A. Hjorth, W. Wienen, M. Entzeroth, ¨ D.J. Bergsma, T.W. Schwartz, Mutations in transmembrane segment VII of the AT1 receptor differentiate between closely related insurmountable and competitive angiotensin antagonists, Br. J. Pharmacol. 113 Ž1994. 331–333. w19x Y. Yamano, K. Ohyama, S. Chaki, D.-F. Guo, T. Inagami, Identification of amino acid residues of rat angiotensin II receptor for ligand binding by site directed mutagenesis, Biochem. Biophys. Res. Commun. 187 Ž1992. 1426–1431. w20x Y. Yamano, K. Ohyama, M. Kikyo, T. Sano, Y. Nakagomi, Y. Inoue, N. Nakamura, I. Morishima, D.F. Guo, T. Hamakubo, T. Inagami, Mutagenesis and the molecular modeling of the rat angiotensin II receptor ŽAT1 ., J. Biol. Chem. 270 Ž1995. 14024–14030. w21x D.K. Yee, P. He, X.-D. Yang, L.P. Reagan, J. Hines, I.R. Siemens, S.J. Fluharty, Cloning and expression of angiotensin II type 2 ŽAT2 . receptors from murine neuroblastoma N1E-115 cells: evidence for AT2 receptor heterogeneity, Mol. Brain Res. 45 Ž1997. 108–116. w22x D.K. Yee, L.R. Kisley, J.N. Heerding, S.J. Fluharty, Mutation of a conserved fifth transmembrane domain lysine residue ŽLys 215 . attenuates ligand binding in the angiotensin II type 2 receptor, Mol. Brain Res. 51 Ž1997. 238–241.
Short communication
Role of the amino terminus in ligand binding for the angiotensin II type 2 receptor Daniel K. Yee, Jennifer N. Heerding, Marc Z. Krichavsky, Steven J. Fluharty
)
Departments of Animal Biology, Pharmacology, Psychology, and the Institute of Neurological Sciences, UniÕersity of PennsylÕania, Philadelphia, PA 19104, USA Accepted 7 April 1998
Abstract Key amino terminal residues in type 1 ŽAT1 . angiotensin II ŽAngII. receptors are not conserved within type 2 ŽAT2 . receptors. We therefore characterized amino terminal mutants that are transiently expressed in COS-3 membranes. AT2 amino terminal deletion drastically reduced affinity for AngII, suggesting its importance for this subtype. AT1 –AT2 amino terminal exchanges retained wild type AngII affinities Ž K d ranging from 3–5 nM., indicating compensation despite substantial sequence dissimilarities. Finally, binding of AT2 selective ligands ŽCGP42112A and PD123319. was not dependent on the amino terminus. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Angiotensin; Amino terminus; Mutagenesis; Ligand binding
Angiotensin II ŽAngII. is one of the key hormones involved in the regulation of cardiovascular and body fluid homeostasis. This peptide hormone affects numerous peripheral target organs, as well as discrete areas of the brain, through its interactions with cell surface receptors at these locations w15x. At least two main families of AngII receptors, referred to as type 1 ŽAT1 . and type 2 ŽAT2 ., have been identified through the use of subtype selective ligands w3x. With the cloning of these AngII receptor subtypes, more detailed structural information on these receptors has become available w11–13,16x. Both subtypes conform to the seven transmembrane-spanning structural motif of G-protein coupled receptors. Mutagenesis of G-protein coupled receptors has yielded valuable insights into the structural and molecular properties of this superfamily of receptors. With respect to AngII receptors, these studies have focused primarily on the AT1 receptor subtype and have identified several structural components that are involved in ligand binding and receptor function w2,8,10,14,17–20x. In contrast to the accumulating data on the structural and molecular properties of the
) Corresponding author. Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104. Fax: q1-215-898-0899; E-mail: [email protected]
0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 1 0 4 - 1
AT1 receptor, the AT2 receptor has been largely neglected with respect to structural analysis by either mutagenesis or molecular modeling. Both the AT1 and AT2 receptors bind AngII with nearly identical affinities, despite sharing only a 34% homology. Given the relatively low level of homology between the AT1 and AT2 receptors, it seems plausible that areas of highest homology may confer AngII binding specificity to the two receptor subtypes. Indeed, a comparison of the amino acid sequences show that some of the key residues identified by AT1 mutagenesis experiments as critical for AngII binding are shared by the two receptor subtypes w8,20x. However, there are other identified AT1 residues that are not conserved in the AT2 subtype. One such area is the amino terminus, which is highly divergent in the two receptor subtypes. Moreover, the AT2 amino terminus is almost twice the size of the analogous AT1 region Žsee Fig. 1.. The importance of this protein domain for high affinity AngII binding in the AT1 subtype has been clearly established by analysis of a series of point mutations in the AT1 amino terminus w8x. However, these critical AT1 residues identified by mutagenesis studies are not conserved in the AT2 receptor Žsee Fig. 1. and therefore high affinity binding to AngII in this receptor family cannot be solely explained by the actions of a few conserved residues in the two subtypes. Since there is currently little information on
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Fig. 1. Alignment of amino acid sequences of AT1 and AT2 amino terminal domains. The amino acid sequences of mouse AT1A and AT2 receptors are compared over their amino terminal regions. Skipped sequences as indicated by hyphens are introduced for maximizing sequence homology. Vertical lines Ž<. between the sequences highlight identical residues between the two subtypes. Previous mutagenesis experiments of the AT1 receptor by Hjorth ¨ et al. w8x have identified several residues that appear to be involved in the binding of AngII. These key AT1 residues are indicated above with asterisks Ž)..
the binding epitopes for the AT2 receptor, any possible contribution towards ligand binding by its amino terminus remains to be established. Consequently, in this paper, we have constructed and characterized an amino terminal deletion of the AT2 receptor subtype as well as generated and analyzed the binding properties of chimeric exchanges of this protein domain between the two receptor subtypes Žsee Fig. 2.. The polymerase chain reaction ŽPCR. was used to create all mutational constructs with AT1 and AT2 receptor cDNAs that have previously been isolated from the murine neuroblastoma N1E-115 cell line w6,21x serving as templates. To construct the AT2 amino terminal deletion delŽ5–40.AT2 , a specially designed forward primer 5XGCGGGATCCAAAATGAAAGATAATTTGGAAGCAATTCCTGTTCTCTAC-3X was employed that preserved the first four amino acids, while removing the following 36 amino acids of the AT2 amino terminus ŽMet-Lys-AspAsn-rest of AT2 receptor.. A complete removal of the amino terminus was not employed because of the concern that such an extreme deletion may interfere with translocation of the mutant receptors to the plasma membrane. Thus, the AT2 deletion mutant Ždel 5–40.AT2 retains the first four amino acids of the amino terminus with the expectation that this construct would not be as disruptive to normal cellular processing as removal of the entire protein domain Žsee Fig. 2.. The amino terminal exchange mutants were generated by the splicing by overlap extension ŽSOE. process w9x. In attaching the AT2 amino terminus onto the AT1 receptor, the overlapping primers 5X-CATGACAAAATGCTTATCTGATGGTTTGTG-3X and 5X-TCAGATAAGCATTTTGTCATGATCCCTACTCTC-3X were used in the SOE reaction. In constructing the AT1 amino terminus onto an AT2 receptor the overlapping primers 5X-TGCTTCCAATATGTAACTGTGCCTGCCAGC-3X and 5X-CACAGTTACATATTGGAAGCAATTCCTGTTCTC-3X were used. A small amount of Pfu, a thermostable, new generation DNA polymerase was also added to the polymerase chain reactions Ž1:100 Pfu:Taq. in order to improve the fidelity of the SOE process w1,4x.
DNA sequencing of all mutant receptors was performed to verify that the desired mutations was produced without any inadvertant misincorporations due to PCR. Following verification, the mutant receptors were then directly ligated into the eukaryotic expression vector, PCR3 ŽInvitrogen.. Transfection of COS-3 cells were performed as previously described w22x. Subsequently, cell membranes were prepared from transfected cells and radioligand binding analysis was performed using monoiodinated 125 I-AngII ŽNENrDupont. as previously described w5,22x. Binding data was then analyzed by the program InPlot ŽGraphPad Software.. The necessity of the AT1 amino terminus for high affinity AngII binding has been previously established by conservative segment exchange and point mutations for this subtype w8x. In contrast, the role of the AT2 amino terminus, which is greatly divergent compared to the AT1 amino terminus, remains to be established. Consequently, in order to investigate a possible role of this protein domain in the AT2 subtype, the deletion mutant Ždel 5–40.AT2 was constructed that removed a large portion of the AT2 amino terminus. This mutant specifically bound 125 I-AngII, indicating that despite deletions of a large portion of the amino terminus, the mutant receptor protein was expressed and properly oriented in the plasma membrane. However, in marked contrast to the wild type receptor Ž K d s 3.78 " 0.15 nM; mean " S.E.M.; n s 3., 125 I-AngII binding was not saturable in the deletion mutant
Fig. 2. Creation of AT2 amino terminal deletion mutant and AT1 –AT2 amino terminal exchange mutants. To address the possible role of the amino terminus in ligand binding for AngII receptors, a deletion mutant for the AT2 amino terminus, as well as chimeric exchange of these protein domains between the AT1 and AT2 subtypes were constructed using the splicing by overlap method. These mutants are depicted above.
D.K. Yee et al.r Molecular Brain Research 57 (1998) 325–329
over the range of ligand concentration that was used Ždata not shown, n s 3.. Although the nonsaturable nature of the mutant binding does not permit a precise determination of receptor expression and ligand affinity by curve-fitting, this nonetheless indicates a drastic reduction in the affinity of the mutant receptor for the ligand Ž K d ) 100 nM, the limits of the curve fitting software over the concentration range of the ligands used.. Therefore, these data suggest that portions of the AT2 amino terminus are required to maintain high affinity binding of AngII. However, since the critical AT1 amino terminal residues are not conserved in the AT2 subtype, the AT2 amino terminus must interact with its ligand via an alternate binding mechanismŽs., i.e., through a different set of amino acid residues. To further investigate the role of the amino termini in the AngII receptor family, we constructed chimeric exchanges of this protein region between the two subtypes Žsee Fig. 2.. Saturation isotherms using 125 I-AngII were subsequently performed on the harvested membrane preparations from transfected COS-3 cells. Although receptor expression varied with transfection efficiency for each of the constructs, the binding for 125 I-AngII was saturable for each of the chimeric receptor and was thus amenable to analysis by the curve fitting software InPlot Žfrom GraphPad.. Bmax values ranged from 1333 to 1677 fmolrmg protein for the AT2 receptor, from 135.1 to 5482 fmolrmg protein for the AT1 receptor, from 37.7 to 261.5 fmolrmg protein for the chimeric AT2 receptor with a substituted AT1 amino terminus ŽAT1r2 NT., and from 8.94 to 29.7 fmolrmg protein for the chimeric AT1 receptor with a substituted AT2 amino terminus ŽAT2r1 NT.. The 125 IAngII binding affinities for the chimeric receptors were essentially identical to the wild type receptors Žunpaired t-test, p - 0.05.: wild type AT2 had a K d of 3.78 " 0.15 nM Ž n s 3. while that of AT1r2 NT was 5.06 " 2.49 Ž n s 3.; wild type AT1 had a K d of 3.23 " 1.78 nM Žmean " S.E.M.; n s 3. while that of AT2r1 NT was 3.26 " 1.17 nM Ž n s 3.. Since the importance of the amino termini in the binding of AngII is established for both subtypes and the structural dissimilarities between the two amino termini are so prominent, it is particularly striking that the chimeric exchanges of this protein domain among the two receptor subtypes retain wild type binding for AngII. These results suggest that although the two receptor subtypes may employ differing amino acid residues in the amino terminus to interact with AngII, these alternate binding mechanismŽs. can compensate for each other, most likely by interacting with a similar portion of the AngII molecule. It is important to note, however, that the loss-of-function data in previous AT1 mutagenesis experiments may simply have reflected global changes in receptor folding that perturbed AngII binding rather than the actual removal of specific binding epitopes in the AT1 amino terminus w8x Žalso see Fig. 1.. Although scanning alanine mutations successfully mapped the location of key AT1 residues in the amino
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terminus through the loss of peptidic binding and is therefore suggestive of the importance of these residues in ligand binding, this strategy cannot specify the nature of the interactions between these targeted residues and the ligand. Consequently, any conclusions drawn from these AT1 experiments must be cautiously considered and an alternate explanation for our chimeric receptor data is that despite the great dissimilarities in their sequences, the exchange of the native amino terminal regions of the AT1 and AT2 receptors could simply be less globally disruptive than the other mutations in previous AT1 experiments. Still, the retention of wild type binding by the chimeric receptors indicate that the amino terminus of one receptor subtype is not incompatible with the remaining protein molecule of the reciprocal subtype and therefore makes it likely that despite the low relative homology between the two subtypes, AngII is situated in the binding pocket of both subtype receptors in a similar general orientation. In their transmembrane domains and in other extracellular domains, the AT1 and AT2 receptors do share some conserved residues. Our previous mutational work on these conserved residues in the AT2 receptor have indicated that some, but not all of these conserved residues may play a role in the binding of AngII w7,22x. These earlier results suggested that despite the relatively low homology, the two AngII receptor subtypes may share some essential commonalities in the manner that they bind AngII and its analogs. Because several of the amino acid residues previously implicated in the binding of AngII by AT1 mutagenesis studies w8,20x are not conserved in the AT2 receptor, the AngII binding mechanisms for both subtypes cannot be entirely identical. As demonstrated by the amino terminal chimeric exchange experiments, other alternate mechanisms that are distinct to each subtype are also necessary and may have developed from the ongoing divergence of these two subtypes during evolution to maintain high affinity for AngII. In addition to investigating the binding of these receptor subtypes to an identical ligand, namely AngII, the amino terminal chimeric exchanges offered an opportunity to map the binding epitopes of subtype specific compounds. With respect to the AT1 receptor, the binding epitopes of its specific antagonist losartan have been extensively investigated and have been determined to reside exclusively within the transmembrane regions of the receptor w10,17,18x. In contrast, a similar understanding of binding of AT2 selective compounds for their subtype remains to be determined. In this regard, the chimeric exchange of the AT1 amino terminus onto an AT2 receptor ŽAT1r2 NT. provided an opportunity to investigate any possible contributions by this protein domain towards the binding of AT2 selective compounds. Therefore, competition binding analysis was performed comparing the AT1r2 NT mutant to the wild type AT2 receptor with respect to both AT1 and AT2 specific ligands. As shown in Fig. 3, replacement of the AT2 amino terminus with that of the AT1 did not
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Fig. 3. Competition of 125 I-AngII binding with various ligands in wild type AT2 and in chimeric AT2 receptor with a substituted AT1 amino terminus. Competition of 0.6 nM 125 I-AngII to wild type AT2 and to chimeric AT1r 2 amino terminal exchange mutant was determined in the presence of increasing concentrations from 10y1 1 to 10y6 M of AngII, losartan, CGP42112A, and PD123319. The competition curves shown above are representative of three independent experiments.
confer losartan binding to this chimeric receptor, confirming that the binding epitopes for this AT1 antagonist are not in the amino terminus. However, 125 I-AngII continued to be effectively displaced by AngII and the AT2 specific compounds CGP42112A and PD123319 for the AT1r2 NT mutant. The K i values for wild type AT2 receptor with respect to AngII, CGP42112A, and PD123319 were respectively 11.0 " 1.68 nM, 3.58 " 0.74 nM, and 19.7 " 2.97 nM Ž n s 4.; while for the chimeric receptor, they were respectively 2.0 " 0.20 nM, 0.695 " 0.083 nM, and 8.99 " 0.60 nM Ž n s 3.. Because small, non-peptide ligands tend to be seated more deeply than larger, peptidic compounds in the ligand binding pocket where residues of the transmembrane domains are primarily responsible for determining binding affinity and specificity, high affinity binding of the non-peptide compound PD123319 by the chimeric receptor was not surprising. On the other hand, high affinity binding of the larger, peptidic CGP42112A
was also preserved in the chimeric receptor, indicating that the binding epitopes that determine AT2 specificity of this ligand do not reside in the amino terminus. Initially, one would have expected that the peptidic CGP42112A would interact with the extracellular domains, including the amino terminus. It is possible that the amino terminus may still contribute towards the binding of CGP42112A, but because it is an analog of AngII this protein region may interact with a portion of CGP42112A that does not confer its subtype specificity. Thus, the chimeric mutant of an AT1 amino terminus onto an AT2 receptor may continue to displace 125 I-AngII with either AngII or CGP42112A with high efficiency. Although the rank order potency for AngII, CGP42112A, and PD123319 was identical for both receptors, these competitors did demonstrate greater efficiency in displacing 125 I-AngII in the chimeric receptor compared to the wild type receptor Žunpaired t-test, p - 0.05.. It is possible that the exchange of the long AT2 amino terminus with the shorter AT1 portion allowed for greater accessibility to the binding pocket for each of these ligands. The use of molecular biological techniques to introduce specific mutations into proteins has proven to be an invaluable research strategy in the analysis of protein structure and function. With respect to the AT1 receptor, these techniques have yielded a growing data set which have been used to define the ligand binding pocket for this receptor subtype. The experiments presented in this paper represent our continuing attempts to extend these approaches to the AT2 receptor subtype. These experiments on the amino terminal regions, a protein domain that greatly differs in the two subtypes, demonstrate that identification of a function, in this case, the high affinity binding of AngII, cannot simply be an inevitable consequence of sequence homology. In fact, the preservation of high affinity binding of AngII by the chimeric receptors indicate that the amino termini of AT1 and AT2 are able to functionally compensate for one another despite great structural and sequence dissimilarities. Yet both subtypes do have some residues in common in other parts of these receptor proteins, which may represent some structural commonalities in the AngII receptor family. Indeed, our initial mutagenesis studies of extracellular and transmembrane domains of the AT2 receptor have suggested that the two AngII receptor subtypes may share some structural commonalities in their binding mechanismŽs. for AngII w7,22x. Thus, evaluating structural similarities and differences between the two receptor subtypes should provide further insights towards understanding the molecular mechanisms that define ligand binding for the entire AngII receptor family.
Acknowledgements This work was supported by NS23986, MH43787, and by Grants-In-Aid from the American Heart Association ŽNational and Southeastern Pennsylvania Affiliate..
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