Probing the Structure and Function of the Tachykinin Neurokinin-2 Receptor through Biosynthetic Incorporation of Fluorescent Amino Acids at Specific Sites

Abstract

A general method for understanding the mechanisms of ligand recognition and activation of G protein-coupled receptors has been developed. A study of ligand-receptor interactions in the prototypic seven-transmembrane neurokinin-2 receptor (NK2) using this fluorescence-based approach is presented. A fluorescent unnatural amino acid was introduced at known sites into NK2 by suppression of UAG nonsense codons with the aid of a chemically misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. Fluorescence-labeled NK2 mutants containing an unique 3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diaminopropionic acid (NBD-Dap) residue at either site 103, in the first extracellular loop, or 248, in the third cytoplasmic loop, were functionally active. The fluorescent NK2 mutants were investigated by microspectrofluorimetry in a native membrane environment. Intermolecular distances were determined by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist. These distances, calculated by the theory of Förster, permit to fix the ligand in space and define the structure of the receptor in a molecular model for NK2 ligand-receptor interactions. Our data are the first report of the incorporation of a fluorescent unnatural amino acid into a membrane protein in intact cells by the method of nonsense codon suppression, as well as the first measurement of experimental distances between a G protein-coupled receptor and its ligand by FRET. The method presented here can be generally applied to the analysis of spatial relationships in integral membrane proteins such as receptors or channels. A general method for understanding the mechanisms of ligand recognition and activation of G protein-coupled receptors has been developed. A study of ligand-receptor interactions in the prototypic seven-transmembrane neurokinin-2 receptor (NK2) using this fluorescence-based approach is presented. A fluorescent unnatural amino acid was introduced at known sites into NK2 by suppression of UAG nonsense codons with the aid of a chemically misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. Fluorescence-labeled NK2 mutants containing an unique 3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diaminopropionic acid (NBD-Dap) residue at either site 103, in the first extracellular loop, or 248, in the third cytoplasmic loop, were functionally active. The fluorescent NK2 mutants were investigated by microspectrofluorimetry in a native membrane environment. Intermolecular distances were determined by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist. These distances, calculated by the theory of Förster, permit to fix the ligand in space and define the structure of the receptor in a molecular model for NK2 ligand-receptor interactions. Our data are the first report of the incorporation of a fluorescent unnatural amino acid into a membrane protein in intact cells by the method of nonsense codon suppression, as well as the first measurement of experimental distances between a G protein-coupled receptor and its ligand by FRET. The method presented here can be generally applied to the analysis of spatial relationships in integral membrane proteins such as receptors or channels. INTRODUCTIONNeurokinin (tachykinin)-2 receptor (NK2) 1The abbreviations used are: NK2neurokinin-2 receptorNKAneurokinin ATMRtetramethylrhodamineFRETfluorescence resonance energy transferNBD-Dap3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diaminopropionic acidcRNAcapped complementary RNAN-Nvoc-Dap(NBD)2-N-(6-Nitroveratryloxycarbonyl)-I-3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-I-2,3-diaminopropionic acid cyanomethylesterSup-tRNA-NBDyeast suppressor tRNAPheCUA (G20U, A73G) acylated with NBD-DapSup-tRNA-Pheyeast suppressor tRNAPheCUA (G20U, A73G) acylated with phenylalanineCHOChinese hamster ovaryststop. is a member of the G protein-coupled receptors family (1Nakanishi S. Annu. Rev. Neurosci. 1991; 14: 123-136Crossref PubMed Scopus (446) Google Scholar). This class of receptors comprises integral membrane proteins that transmit signals into cells in response to a variety of extracellular stimuli such as hormones of a variety of structures, from monoamines to large peptides, and even light or ions. The particular receptors are predicted to have seven transmembrane regions and are believed to interchange between a number of different conformations that can selectively bind agonist or antagonist ligands and activate G proteins with important functional consequences (2Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 3Rosenkilde M.M. Cahir M. Gether U. Hjorth S.A. Schwartz T.W. J. Biol. Chem. 1994; 269: 28160-28164Abstract Full Text PDF PubMed Google Scholar). G protein-coupled receptors exploit diverse strategies for ligand recognition, using either the transmembrane domain, the extracellular surface, or even the N-terminal segment. Detailed structural information is important to the understanding of the mechanisms of ligand recognition and activation of G protein-coupled receptors and in the design of therapeutic agents. Obtaining insight into the three-dimensional structure of these receptors represents a formidable task because of the difficulties of obtaining and purifying sufficiently large amounts of protein to produce crystals for high resolution x-ray or electron diffraction. Novel biochemical and biophysical methods are needed not only to overcome the hurdle to explore the molecular architecture of receptors (4Hubbell W.L. Altenbach C. Curr. Opin. Struct. Biol. 1994; 4: 566-573Crossref Scopus (359) Google Scholar, 5Farahbakhsh Z.T. Ridge K.D. Khorana H.G. Hubbell W.L. Biochemistry. 1995; 34: 8812-8819Crossref PubMed Scopus (185) Google Scholar) but also for investigating the dynamics of their interaction with ligands.One powerful approach is the investigation of extrinsic fluorophores incorporated at known sites in the receptor or ligand by fluorescence spectroscopy. Fluorescent labels serve as sensitive indicators of the microenvironment at a given residue as well as structural changes in receptor (6Turcatti G. Vogel H. Chollet A. Biochemistry. 1995; 34: 3972-3980Crossref PubMed Scopus (54) Google Scholar, 7Fay S.P. Domalewski M.D. Sklar L.A. Biochemistry. 1993; 32: 1627-1631Crossref PubMed Scopus (22) Google Scholar, 8Gether U. Lin S. Kobilka B.K. J. Biol. Chem. 1995; 270: 28268-28275Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Time-resolved techniques will permit the study of the structural changes to which a given residue might be submitted during receptor activation in a native membrane environment. Finally, fluorescence resonance energy transfer between a donor and acceptor pair located in the receptor and the ligand will permit intermolecular distances between these labels to be estimated.Introducing fluorescent groups at specific sites in G protein-coupled receptors presents a major challenge. Classical methods such as solid-phase synthesis or chemical modification of reactive side chains have been used in vitro to incorporate unnatural biophysical probes into proteins but few of these approaches are suitable for large membrane proteins (9Ridge K.D. Zhang C. Khorana H.G. Biochemistry. 1995; 34: 8804-8811Crossref PubMed Scopus (40) Google Scholar). In the recent years, expansion of the genetic code by suppression of the nonsense stop codon UAG has been developed for the site-directed incorporation of a variety of unnatural amino acids with novel properties into proteins in vitro in cell-free systems (10Cornish V.W. Mendel D. Schultz P.G. Angew. Chem. Int. Ed. Engl. 1995; 34: 621-633Crossref Scopus (233) Google Scholar, 11Bain J.D. Diala E.S. Glabe C.G. Wacker D.A. Lyttle M.H. Dix T.A. Chamberlin A.R. Biochemistry. 1991; 30: 5411-5421Crossref PubMed Scopus (101) Google Scholar, 12Noren C.J. Anthony-Cahill S.J. Griffith M.C. Schultz P.G. Science. 1989; 244: 182-188Crossref PubMed Scopus (859) Google Scholar). Only recently has this been extended to heterologous expression of membrane proteins in an intact cell system: the Xenopus oocyte (13Nowak M.W. Kearney P.C. Sampson J.R. Saks M.E. Labarca C.G. Silverman S.K. Zhong W. Thorson J. Abelson J.N. Davidson N. Schultz P.G. Dougherty D.A. Lester H.A. Science. 1995; 268: 439-442Crossref PubMed Scopus (213) Google Scholar).Here we report a novel biophysical approach for understanding the structure and function of a representative G protein-coupled receptor, the tachykinin NK2 receptor. This approach combines the biosynthetic incorporation of extrinsic fluorescent reporter groups into proteins in intact cells with the widely employed technique of fluorescence resonance energy transfer (FRET) (14Förster T. Ann. Physiol. (Leipzig). 1948; 2: 55-75Crossref Scopus (4844) Google Scholar, 15Stryer L. Annu. Rev. Biochem. 1978; 47: 819-846Crossref PubMed Scopus (1952) Google Scholar) for distance measurements. Here we use these experimentally determined distances to build a model for the interaction of a peptide ligand with NK2 in a native membrane environment.DISCUSSIONIn this study we present a novel fluorescence spectroscopic approach to obtain structural information on integral membrane proteins as demonstrated for the NK2 neurokinin G protein-coupled receptor. The aim was to use FRET techniques between a fluorescent donor group engineered in the NK2 receptor at known sites, and an acceptor fluorophore in the ligand, for the determination of a structural model for interaction of a peptide ligand with NK2.Site-specific fluorescent labeling of NK2 was accomplished in intact cells by suppression of TAG nonsense codon placed at known sites in NK2 using a suppressor tRNA aminoacylated with the fluorescent L-amino acid analogue NBD-Dap. The key steps in this approach are: 1) the mutation of a selected site in NK2 cDNA to TAG termination codon and the preparation of the cognate cRNA transcript containing the UAG codon; 2) the construction by chemical and enzymatic methods of a misacylated suppressor tRNA that recognize the UAG stop codon and functions biosynthetically as a source of unnatural amino acid; and 3) the heterologous expression of receptor containing unnatural amino acids in Xenopus oocytes. The oocyte cell translation machinery recognizes the exogeneous aminoacylated suppressor tRNA and incorporates the fluorescent residue into the nascent protein at sites preselected by creation of a nonsense codon in the gene. To improve the efficiency of the incorporation, we designed and constructed a synthetic UAG suppressor tRNA (Sup-tRNA-NBD, Fig. 1) derived from yeast tRNA(Phe). This contained the anticodon CUA that recognizes the UAG stop mutation in the cRNA. The fluorescent amino acid NBD-Dap was acylated chemically to the 3′-end and two mutations (G20U and A73G) were introduced to reduce the rate of reacylation with natural amino acids by endogeneous aminoacyl-tRNA synthetases (28Bruce A.G. Atkins J.F. Wills N. Uhlenbeck O. Gesteland R.F. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7127-7131Crossref PubMed Scopus (25) Google Scholar, 29Sampson J.R. DiRenzo A.B. Behlen L.S. Uhlenbeck O.C. Science. 1989; 243: 1363-1366Crossref PubMed Scopus (148) Google Scholar). UAG was chosen as a stop codon for controlled suppression because of its low usage as a natural termination codon in oocytes (17%), compared to UAA (36%) or UGA (47%) (32Cherry J.M. Methods Cell Biol. 1991; 36: 675-677Crossref PubMed Scopus (3) Google Scholar). The yields of suppression were in the 10-30% range. We never detected NBD fluorescence in membranes prepared from oocytes injected with the fluorescent Sup-tRNA-NBD only. This suggests that suppression of UAG stop codons naturally occurring in oocytes by the exogeneously added suppressor tRNA was insignificant in the context of this study.The functional activity of NK2 and fluorescent suppression mutants (Fig. 2) was assayed by activation of Ca2+-dependent chloride currents (24Nemeth K. Chollet A. J. Biol. Chem. 1995; 270: 27601-27605Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 30Harada Y. Takahashi Kuno M. Nakyama K. Masu Y. Nakanishi S. J. Neurosci. 1987; 7: 3265-3273Crossref PubMed Google Scholar). The minor differences observed among the chloride currents profiles represent the degree of variability of response that is normally seen in oocytes. Similar oocyte to oocyte variations were observed for wild type NK2 in the same batch. The data demonstrate that functional NK2 mutant receptors, containing the unnatural NBD-Dap amino acid at either position 103 or 248, were synthesized by oocytes. The only detectable difference between suppression mutants and wild type receptor was the latency of onset of chloride channel opening (Fig. 3) which probably reflects the differences in expression level.Fluorescent NK2 receptors in oocyte membranes were further characterized by saturation binding analysis with the specific NK2 antagonist PhCO-(δ-125I-phenol-3-propionyl)-Orn-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2. Kd values for binding of this ligand to oocyte membrane fractions were comparable for wild-type NK2 and R103NBD and F248NBD mutants. Moreover, these Kd values were comparable to those obtained for NK2 receptor stably expressed in CHO cells (Table I), thus indicating that the structural integrity of the mutants was conserved.Fluorescent labels placed at known sites can monitor conformational changes in receptors in response to ligand binding. In that respect, position 248 in the C-terminal part of the third intracellular loop of NK2 is particularly interesting as this region has been shown to be involved in receptor activation and G protein coupling for other 7-transmembrane receptors (38Burstein E.S. Spalding T.A. Hill-Eubanks D. Brann M.R. J. Biol. Chem. 1995; 270: 3141-3146Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 39Liu J. Conklin B.R. Blin N. Yun J. Wess J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11642-11646Crossref PubMed Scopus (195) Google Scholar). We observed no change in the fluorescence of mutant F248NBD during binding of either agonist or antagonist (Fig. 5). Without totally ruling out that this region undergoes conformational change during receptor functioning, this however indicates that there is no detectable change in the hydrophobicity around site 248 because NBD fluorescence depends strongly on the polarity of its environment.Evidence for fluorescence energy transfer was provided by quenching of the NBD-labeled receptor (donor) fluorescence. This quenching was used to quantify the energy transfer because, under the conditions of measurement, there was no evidence for other deactivation process of NBD fluorescence. An important factor influencing the FRET experiments and their interpretation is the dipole orientation factor κ2. In theory, this factor can take any value between 0 and 4. κ2 can be precisely determined only if the positions of the two fluorescent molecules is fixed and known. When the energy donor is free to rotate and the energy acceptor is fixed, κ2 can range from 1/3 compared to 4/3 depending on the angle between the transition moment of the acceptor and the donor-acceptor separation vector (15Stryer L. Annu. Rev. Biochem. 1978; 47: 819-846Crossref PubMed Scopus (1952) Google Scholar). The error on the calculated R0 due to the uncertainty in the value of κ2 in this range is ±11%. We are making this assumption in the experiments described in this study. From the steady state fluorescence anisotropy experiments, we can deduce that the TMR-labeled ligand bound to NK2 is immobilized. Also it is reasonable to assume that residues 103 and 248 located in loops connecting membrane-spanning domains have some freedom to rotate.In previous spectrofluorometric observations and collisional quenching experiments from our laboratory (6Turcatti G. Vogel H. Chollet A. Biochemistry. 1995; 34: 3972-3980Crossref PubMed Scopus (54) Google Scholar) we have shown that the antagonist heptapeptide ligand binds the wild type receptor in a hydrophobic pocket on the extracellular side of the transmembrane segments and that the fluorophore is located at the membrane-water interface. The distance between the bound TMR ligand and position 248 estimated by FRET is consistent with a seven transmembrane topology for the NK2 receptor and provides an experimental validation of this model. The membrane bilayer can be viewed as a hydrophobic core region of about 30 Å flanked by two interfacial regions of about 15 Å each (40White S.H. Wimley W.C. Curr. Opin. Struct. Biol. 1994; 4: 79-86Crossref Scopus (170) Google Scholar). The acceptor and donor fluorophores are predicted in the interfacial areas on opposite sides of the membrane. Residues Gln109, Cys167, His198, and Gly273 around the extracellular side of the third, fourth, fifth, and transmembrane were to be for peptide antagonist binding to D. and J. C. submitted for the NK2 residue 248 on the cytoplasmic residue 103 in the first extracellular loop, and into the FRET distances with the TMR the position of the ligand in This in the three-dimensional model of NK2 in Fig. using the structure of as a general for the G protein-coupled receptors G. C. 1993; PubMed Scopus Google Scholar). To this we the FRET experimental distances with the other Fig. the orientation for the fluorescent heptapeptide antagonist bound to in the context of this model. The fluorescent TMR group of the by the seven membrane of the peptide ligand between the and transmembrane domains suggests that be by of the for receptor measurement of FRET distances between the fluorescent ligand, or an analogue labeled at a different and other labeled sites in NK2 will permit further of this we presented the first report of the of fluorescent probes at specific sites in a membrane receptor in intact cells by unnatural suppression We also that these labeled receptors can be used to intermolecular distances between these sites and bound the fluorescent to different sites in the receptor, it will be to improve the structural model of the receptor and its ligands in a native membrane environment. In the fluorescent will permit the study of changes in the receptor structure J.R. W. G. Chem. 36: PubMed Scopus Google Scholar, H. L. S. G. W. G. J. Biochem. 1993; PubMed Scopus Google Scholar). These methods can be generally applied to the molecular architecture and conformational changes of integral membrane proteins such as or receptors as well as to or interactions. INTRODUCTIONNeurokinin (tachykinin)-2 receptor (NK2) 1The abbreviations used are: NK2neurokinin-2 receptorNKAneurokinin ATMRtetramethylrhodamineFRETfluorescence resonance energy transferNBD-Dap3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-diaminopropionic acidcRNAcapped complementary RNAN-Nvoc-Dap(NBD)2-N-(6-Nitroveratryloxycarbonyl)-I-3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-I-2,3-diaminopropionic acid cyanomethylesterSup-tRNA-NBDyeast suppressor tRNAPheCUA (G20U, A73G) acylated with NBD-DapSup-tRNA-Pheyeast suppressor tRNAPheCUA (G20U, A73G) acylated with phenylalanineCHOChinese hamster ovaryststop. is a member of the G protein-coupled receptors family (1Nakanishi S. Annu. Rev. Neurosci. 1991; 14: 123-136Crossref PubMed Scopus (446) Google Scholar). This class of receptors comprises integral membrane proteins that transmit signals into cells in response to a variety of extracellular stimuli such as hormones of a variety of structures, from monoamines to large peptides, and even light or ions. The particular receptors are predicted to have seven transmembrane regions and are believed to interchange between a number of different conformations that can selectively bind agonist or antagonist ligands and activate G proteins with important functional consequences (2Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 3Rosenkilde M.M. Cahir M. Gether U. Hjorth S.A. Schwartz T.W. J. Biol. Chem. 1994; 269: 28160-28164Abstract Full Text PDF PubMed Google Scholar). G protein-coupled receptors exploit diverse strategies for ligand recognition, using either the transmembrane domain, the extracellular surface, or even the N-terminal segment. Detailed structural information is important to the understanding of the mechanisms of ligand recognition and activation of G protein-coupled receptors and in the design of therapeutic agents. Obtaining insight into the three-dimensional structure of these receptors represents a formidable task because of the difficulties of obtaining and purifying sufficiently large amounts of protein to produce crystals for high resolution x-ray or electron diffraction. Novel biochemical and biophysical methods are needed not only to overcome the hurdle to explore the molecular architecture of receptors (4Hubbell W.L. Altenbach C. Curr. Opin. Struct. Biol. 1994; 4: 566-573Crossref Scopus (359) Google Scholar, 5Farahbakhsh Z.T. Ridge K.D. Khorana H.G. Hubbell W.L. Biochemistry. 1995; 34: 8812-8819Crossref PubMed Scopus (185) Google Scholar) but also for investigating the dynamics of their interaction with ligands.One powerful approach is the investigation of extrinsic fluorophores incorporated at known sites in the receptor or ligand by fluorescence spectroscopy. Fluorescent labels serve as sensitive indicators of the microenvironment at a given residue as well as structural changes in receptor (6Turcatti G. Vogel H. Chollet A. Biochemistry. 1995; 34: 3972-3980Crossref PubMed Scopus (54) Google Scholar, 7Fay S.P. Domalewski M.D. Sklar L.A. Biochemistry. 1993; 32: 1627-1631Crossref PubMed Scopus (22) Google Scholar, 8Gether U. Lin S. Kobilka B.K. J. Biol. Chem. 1995; 270: 28268-28275Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Time-resolved techniques will permit the study of the structural changes to which a given residue might be submitted during receptor activation in a native membrane environment. Finally, fluorescence resonance energy transfer between a donor and acceptor pair located in the receptor and the ligand will permit intermolecular distances between these labels to be estimated.Introducing fluorescent groups at specific sites in G protein-coupled receptors presents a major challenge. Classical methods such as solid-phase synthesis or chemical modification of reactive side chains have been used in vitro to incorporate unnatural biophysical probes into proteins but few of these approaches are suitable for large membrane proteins (9Ridge K.D. Zhang C. Khorana H.G. Biochemistry. 1995; 34: 8804-8811Crossref PubMed Scopus (40) Google Scholar). In the recent years, expansion of the genetic code by suppression of the nonsense stop codon UAG has been developed for the site-directed incorporation of a variety of unnatural amino acids with novel properties into proteins in vitro in cell-free systems (10Cornish V.W. Mendel D. Schultz P.G. Angew. Chem. Int. Ed. Engl. 1995; 34: 621-633Crossref Scopus (233) Google Scholar, 11Bain J.D. Diala E.S. Glabe C.G. Wacker D.A. Lyttle M.H. Dix T.A. Chamberlin A.R. Biochemistry. 1991; 30: 5411-5421Crossref PubMed Scopus (101) Google Scholar, 12Noren C.J. Anthony-Cahill S.J. Griffith M.C. Schultz P.G. Science. 1989; 244: 182-188Crossref PubMed Scopus (859) Google Scholar). Only recently has this been extended to heterologous expression of membrane proteins in an intact cell system: the Xenopus oocyte (13Nowak M.W. Kearney P.C. Sampson J.R. Saks M.E. Labarca C.G. Silverman S.K. Zhong W. Thorson J. Abelson J.N. Davidson N. Schultz P.G. Dougherty D.A. Lester H.A. Science. 1995; 268: 439-442Crossref PubMed Scopus (213) Google Scholar).Here we report a novel biophysical approach for understanding the structure and function of a representative G protein-coupled receptor, the tachykinin NK2 receptor. This approach combines the biosynthetic incorporation of extrinsic fluorescent reporter groups into proteins in intact cells with the widely employed technique of fluorescence resonance energy transfer (FRET) (14Förster T. Ann. Physiol. (Leipzig). 1948; 2: 55-75Crossref Scopus (4844) Google Scholar, 15Stryer L. Annu. Rev. Biochem. 1978; 47: 819-846Crossref PubMed Scopus (1952) Google Scholar) for distance measurements. Here we use these experimentally determined distances to build a model for the interaction of a peptide ligand with NK2 in a native membrane environment.