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Role of the His273 Located in the Sixth Transmembrane Domain of the Angiotensin II Receptor Subtype AT2 in Ligand–Receptor Interaction
Biochemical and Biophysical Research Communications 257, 704 –707 (1999) Article ID bbrc.1999.0207, available online at http://www.idealibrary.com on
Role of the His273 Located in the Sixth Transmembrane Domain of the Angiotensin II Receptor Subtype AT2 in Ligand–Receptor Interaction Cortney A. Turner, Shannon Cooper, and Lakshmidevi Pulakat 1 Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403
Received December 21, 1998
Angiotensin II receptor subtypes AT1 and AT2 are proteins with seven transmembrane domain (TMD) topology and share 34% homology. It was shown that His256, located in the sixth TMD of the AT1 receptor, is needed for the agonist activation by the Phe8 side chain of angiotensin II, although replacing this residue with arginine or glutamine did not significantly alter the affinity binding of the receptor. We hypothesized that the His273 located in the sixth transmembrane domain of the AT2 receptor may play a similar role in the functions of the AT2 receptor, although this residue was not identified as a conserved residue in the initial homology comparisions. Therefore, we replaced His273 of the AT2 receptor with arginine or glutamine and analyzed the ligand-binding properties of the mutant receptors using Xenopus oocytes as an expression system. Our results suggested that the AT2 receptor mutants His273Arg and His273 Glu have lost their affinity to [ 125I-Sar 1-Ile 8]Ang II, a peptidic ligand that binds both the AT1 and AT2 receptors and to 125 I-CGP42112A, a peptidic ligand that binds specifically to the AT2 receptor. Thus, His273 located in the sixth TMD of the AT2 receptor seems to play an important role in determining the binding properties of this receptor. Moreover, these results along with our previous observation that the Lys215 located in the 5th TMD of the AT2 receptor is essential for its high affinity binding to [ 125I-Sar 1-Ile 8]Ang II indicate that key amino acids located in the 5th and 6th TMDs of the AT2 receptor are needed for high affinity binding of the AT2 to its ligands. © 1999 Academic Press
Angiotensin II (Ang II) is an octapeptide hormone that is well known for its role in the regulation of cardiovascular functions and body fluid homeostasis [1–5]. Two major classes of cell surface receptors for Ang II, AT1 and AT2, have been cloned to date [6 –15]. 1
To whom correspondence should be addressed. Fax: (419) 372 2024. E-mail: [email protected]. 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
These receptors have different phamacological properties; the receptor subtype AT1 specifically binds a nonpeptidic ligand losartan (DUP753) and the receptor subtype AT2 specifically binds the peptidic ligand CGP42112A as well as the non-peptide ligand PD123319 [16 –20]. The AT1 receptor is responsible for mediating most of the known physiological effects of Ang II including the vasoconstriction, secretion of aldosterone and antidiuretic hormone and induction of thirst. This receptor is a peptide of 359 amono acids with seven transmembrane domains and belongs to the G-protein coupled-receptor family. The AT1 receptor is shown to activate both Gi and Gq type G-proteins. The AT1 receptor is also known to activate Jak/STAT proteins and mitogen activated protein kinases, and to induce cell proliferation [3, 20]. Detailed mutagenesis and molecular modeling studies have identified the key amino acids of the AT1 receptor involved in ligand binding and G-protein coupling [22–28]. The AT2 receptor is a peptide of 363 amino acids and shares 34% homology with the AT1 receptor. Like the AT1 receptor, the AT2 receptor also has a seven transmembrane domain topology, suggesting that this receptor also belongs to the G-protein coupled-receptor family. However, unlike the AT1 receptor, AT2 does not demonstrate the GTPgS-induced shift to a low affinity form that is characteristic of the G-protein-linked receptors. The AT2 receptor is known to act in a synergistic manner with the AT1 receptor in the activation of phospholipase A 2 and production of arachidonic acid in cardiac ischemia [29]. In contrast, the AT2 receptor is also known to exert opposing effects to that of the AT1 receptor on different cell types, since the AT2 receptor is shown to induce apoptosis whereas the AT1 receptor promotes cell proliferation [30 –32]. Both the AT1 and the AT2 receptor subtypes are often co-expressed [33]. Since these receptors may function in a synergistic manner in certain physiological conditions and in opposing manner in other physiological conditions, it is important to understand the
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FIG. 1. Predicted organization of rat AT2 receptor. Five putative N-linked glycosylation sites are marked (Y). The Histidine residue at position 273 in the sixth transmembrane domain of AT2 receptor that was subjected to mutagenesis is marked by the arrow.
similarities and differences between the structurefunction relationship of these receptors. The amino acid His256 is located in the sixth transmembrane domain of the AT1 receptor. Interaction between the Phe8 of Ang II and this His256 residue is shown to be essential for the agonist activation of the AT1 receptor [22]. The amino acid His273 is located in the sixth transmembrane domain of the AT2 receptor [14, 15]. Although initial comparisons of amino acid sequences of the AT1 and the AT2 receptors did not recognize this Histidine residue as a conserved residue [15], we felt that the location of this residue in the sixth transmembrane domain implies that this residue may play an analogus role to that of His256 of the AT1 receptor regarding the structure-function relationship of the AT2 receptor. Here we report the ligand-binding properties of two mutant AT2 receptors in which the His273 was replaced by glutamine or arginine. MATERIALS AND METHODS Materials. Restriction enzymes were purchased either from Boehringer Mannheim (Indianapolis, IN) or from Promega (Madison, WI). Oligonucleotides used for sequencing and mutagenesis were purchased from GIBCO BRL Life Technologies Inc. (Gaithersbureg, MD). Radiolabeled material for sequencing and binding studies ([ 35S]-dATP, [ 125I-Sar 1-Ile 8]Ang II, 125I-CGP42112A) were obtained from Dupont NEN (Boston, MA). Sequenase Version 2.0 DNA Sequencing Kit was from United States Biochemical, Cleveland, OH
and the DTth DNA Polymerase Sequencing Kit was from ClonTech, Palo Alto, CA. Mutagenesis was performed using Quick Change site-directed mutagenesis kit from Stratagene Products, La Jolla, CA. Riboprobe Gemini in vitro transcription system from Promega, Madison, WI was employed for in vitro transcription of the wild type and mutated AT2 receptors. Xenopus laevis were obtained from Xenopus One (Ann Arbor, MI). Mutagenesis of rat AT2 receptor. The rat AT2 receptor gene cloned in the pSP64 polyA vector [34] was used for the mutagenesis of the His273 codon using the Quick Change site-directed mutagenesis kit. Two synthetic oligonucleotides containing the desired mutation were used as primers [His273 Sense 27mer: 59-TGGCTTCCCTTCC(G/A)GGTTCTGACCTTC-39, His273 Antisense 27mer: 59-GAAGGTCAGAACC(T/C)GGAAGGGAAGCCA-39]. This was because double-stranded DNA was used as the template in this method. The mutated DNA was synthesized by Pfu DNA polymerase using the following thermocycling steps: Denaturing at 95°C for 2 minutes, Annealing at 55°C for 1 minute and then synthesizing at 68°C for 10 minutes. The program was repeated for 17 cycles and then the parental DNA template was digested using DpnI endonuclease which is specific for methylated and hemimethylated DNA. This DNA was then used for transforming E. coli TG1 cells and the mutants were identified by dideoxy nucleotide sequencing using DTth DNA Polymerase Sequencing Kit. In vitro transcription, expression in Xenopus oocytes and binding studies. In vitro transcription of the pSP64 poly(A) vectors carrying the wild type and mutant AT2 receptor genes was carried out using ‘Riboprobe Gemini Systems’ to generate cRNAs corresponding to the wild type and mutant AT2 receptor genes. Techniques for injection of cRNA were performed as described previously [34]. Oocytes were microinjected with 50 nl of cRNA solution at a concentration of 1 mg/ml and after two days the microinjected oocytes were treated
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FIG. 2. Ligand binding properties of the His273Arg and His273Gln mutants. The specific binding of the [ 125I-Sar 1-Ile 8]Ang II (0.5 nM) and I-CGP42112A (0.25 nM) to the oocytes expressing wild-type and mutated receptors are compared. Binding experiments were conducted for a period of one hour according to the procedures described previously [34]. Results are shown as relative % of the binding of the appropriate ligand to the oocytes expressing wildtype AT2 receptor. Receptor expression was quantitated by binding studies using 4 to 7 oocytes from at least 3 donors (a total of at least 12 oocytes). Three different cRNA preparations were used for each sample. Standard errors were with in 3% of each value. 125
with collagenase (0.5 mg/ml) for 20 minutes. The oocytes from each donor frog were screened to ensure that they did not express native Ang II receptors before injecting with AT2 receptor cRNAs by performing binding experiments with [ 125I-Sar 1-Ile 8]Ang II. The binding of [ 125I-Sar 1-Ile 8]Ang II or 125I-CGP42112A to single oocytes was carried out using the procedures described previously [34].
RESULTS AND DISCUSSION Mutagenesis and molecular modeling studies of the AT1 receptor have identified several key residues essential for the functions of this receptor. It has been shown that the amino acids Lys199 (located in the fifth transmembrane domain (TMD) of the AT1 receptor) and His256 (located in the sixth TMD of the AT1 receptor) play important roles in the binding of Ang II and agonist activation of the receptor, respectively [22]. It was also demonstrated that these residues play their role in the binding and activation of the AT1 receptor by directly interacting with the side chain of the Phe8 residue of Ang II. Previous amino acid comparisons between the AT1 and the AT2 receptors identified Lys215 located in the 5th TMD of this receptor as comparable to the Lys199 of the AT1 receptor [15]. Our mutational analysis showed that the Lys215 was essential for the binding of the [ 125I-Sar 1-Ile 8]Ang II to the AT2 receptor [34]. However, replacing this residue with Glutamic acid or Glutamine did not affect the afiinity of the receptor to the AT2 receptor specific peptidic ligand CGP42112A or the non-peptidic ligand PD123319. Thus, it seems that [ 125I-Sar 1-Ile 8]Ang II binding to rat AT2 receptor is comparable to the bind-
ing of this ligand to the rat AT1A receptor. However, binding requirements of the AT2 receptor-specific peptidic ligand, CGP42112A, are not similar to that of [ 125I-Sar 1-Ile 8]Ang II, or other peptidic ligands of AT1 receptor, since replacing Lys215 with Gln or Glu did not impair the affinity of this receptor to CGP42112A. Since His256 located in the sixth TMD was the other key amino acid of the AT1 receptor that directly interacts with the Phe8 of Ang II, and caused agonist activation of the receptor, we analyzed to see if the sixth TMD of the AT2 receptor also contained a Histidine residue that may function in a similar manner to that of His256. We identified His273 located in the sixth transmembrane domain of the AT2 receptor as the putative candidate (Figure 1), although this residue was not identified as a conserved residue in the previous homology comparisons (15). Since replacing the His256 with Arg or Gln did not affect the ligand binding properties of the AT1 receptor considerably [22], we analyzed to see if replacing His 273 by Arg or Gln influenced the ligand-binding properties of the AT2 receptor. To determine the ligand-binding properties of the mutated AT2 receptors, we microinjected Xenopus oocytes with equal amounts of cRNAs encoding the wildtype or the mutated AT2 receptors as described in the Materials and Methods. The follicular layer of the oocytes was removed by collagenase treatment followed by manual removal as described previously [34]. Ligand binding experiments were performed using [ 125ISar 1-Ile 8]Ang II, a peptidic ligand that binds both
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the AT1 and the AT2 receptor subtypes and 125ICGP42112A, a peptidic ligand that binds the AT2 receptor specifically. It was observed that both His273Arg and His273Gln mutants of the AT2 receptor have lost their affinity to [ 125I-Sar 1-Ile 8]Ang II and 125ICGP42112A (Figure 2). Thus His273 located in the sixth TMD of the AT2 receptor seems to be essential for the ability of the receptor to bind its peptidic ligands. Studies using photolabile Ang II analogs have shown that the amino terminal of the AT2 receptor interacts with the aminoterminal end of the Ang II and the the 3rd TMD of the AT2 receptor interacts with the carboxyl-terminal end of the Ang II [35]. Our mutagenesis studies suggest that as in the AT1 receptor, the amino acids located in the region spanning 5th and 6th TMD of the AT2 receptor also play important roles in the binding affinity of the AT2 receptor. Moreover, our results also show that, replacing His273 of the AT2 receptor, a residue analogous to His256, with Arg or Gln, resulted in generating mutants that have lost affinity to two peptidic ligands [ 125I-Sar 1-Ile 8]Ang II and 125I-CGP42112A. Based on these observations we conclude, that the His273 of the AT2 receptor is directly involved in determining the ligand-binding properties of the AT2 receptor. ACKNOWLEDGMENTS We also thank the members of Gavini/Pulakat laboratories at BGSU for their technical help during the course of this work and for helpful discussions. We thank B. Randall and D. Pax for expert animal care. This work is supported by NIH, National Heart, Lung, and Blood Institute Grant, HL60241 to L.P. This work is also supported by Research Challenge Grant, Faculty Research Committee Grant and Faculty Graduate Research Assistantship from BGSU to L.P.
REFERENCES 1. de Gasparo, M., Rogg, H., Brink, M., Wang, L., Whitebread, S., Bullock, G., and Erne, P. (1994) Eur. Heart J. 15(Suppl. D), 98 –103. 2. Dzau, V. J., Mukoyama, M. and Pratt, R. E. (1994) J. Hypertension Suppl. 12, S1–S5. 3. Horiuchi, M. (1996) in Functional Aspects of Angiotensin Type 2 Receptor (Raizada, M. K., Phillips, M. I., and Sumners, C., Eds.), Recent Advances in Cellular and Molecular Aspects of Angiotensin Receptors, pp. 217–224. Plenum Press, New York. 4. Lifton, R. P. (1995) Proc. Natl. Acad. Sci. USA 92, 8545– 8551. 5. Timmermans, P. B. M. W. M., Benfield, P., Chiu, A. T., Herblin, W. F., Wong, P. C., and Smith, R. D. (1992) Am. J. Hypertension 5, 221S–235S. 6. Bergsma, D. J., Ellis, C., Kumar, C., Nuthulaganti, P., Kersten, H., Elshourbagy, N., Griffin, E., Stadel, J. M., and Aiyar, N. (1992) Biochem. Biophys. Res. Commun. 183, 989 –995. 7. Fluharty, S. J., Reagan, L. P., and Yee, D. K. (1995) Adv. Exp. Med. Biol. 377, 193–215. 8. Iwai, N., and Inagami, T. (1992) FEBS Lett. 298, 257–260. 9. Mauzy, C. A., Hwang, O., Egloff, A. M., Wu, L.-H., and Chung, F.-Z. (1992) Biochem. Biophys. Res. Commun. 186, 277–284.
10. Murphy, T. J., Alexander, R. W., Griendling, K., Runge, M. S., and Bernstein, K. E. (1991) Nature 351, 233–236. 11. Sandberg, K., Ji, H., Clark, J. A. L., Shapira, H., and Catt, K. J. (1992) J. Biol. Chem. 267, 9455–9458. 12. Sassaki, K., Yamono, Y., Bardhan, S., Iwai, N., Murray, J. J., Hasegawa, M., Matsuda, Y., and Inagami, T. (1991) Nature 351, 230 –232. 13. Tsuzuki, S., Ichiki, T., Nakakubo, H., Kitami, Y., Guo, D. F., Shirai, H., and Inagami, T. (1994) Biochem. Biophys. Res. Commun. 200, 1449 –1454. 14. Kambayashi, Y., Bardhan, S., Takahashi, K., Tsuzuki, S., Inui, H., Hamakubo, T., and Inagami, T. (1993) J. Biol. Chem. 268, 24543–24546. 15. Mukoyama, M., Nakajima, M., Horiuchi, M., Sasamura, H., Pratt, R. E., and Dzau, V. J. (1993) J. Biol. Chem. 268, 24539 – 24542. 16. Deleted in proof. 17. Chang, R. S. L., and Lotti, V. J. (1991). Life Sci. 49, 1485–1490. 18. Chang, R. S. L., Lotti, V. J. Chen, T. B., Omalley, S. S., Bendesky, R. J., Kling, P. J., Kivlighn, S. D., Siegl, P. K. S., Ondeyka, D., Greenlee, W. J., and Mantlo, N. B. (1995) Eur. J. Pharmacol. 294, 429 – 437. 19. Wexler, R. R., Carini, D. J., Duncia, J. V., Johnson, A. L., Wells, G. J., Chiu, A. T., Wong, P. C., and Timmermanns, P. B. M. W. M. (1992) Am. J. Hypertension 5, 209S–220S. 20. Wexler, R. R., Greenlee, W. J., Irvin, J. D., Goldberg, M. R., Prendergast, K., Smith, R. D., and Timmermans, P. B. M. W. M. (1996) J. Med. Chem. 39, 625– 656. 21. Marrero, M. B., Schieffer, B., Paxton, W. G., Heerdt, L., Berk, B. C., Delafontaine, P., and Bernstein, K. E. (1955) Nature 375, 247–250. 22. Noda, K., Saad, Y., and Karnik, S. S. (1995) J. Biol. Chem. 270, 28511–28514. 23. Noda, K., Saad, Y., Kinoshita, A., Boyle, T., Graham, R., Husain, A., and Karnik, S. S. (1995) J. Biol. Chem. 270, 2284 –2289. 24. Yamano, Y., Ohyama, K., Kikyo, M., Sano, T., Nakagomi, Y., Inoue, Y., Nakamura, N., Morishima, I., Guo, D. F., Hamakubo, T., and Inagami, T. (1995) J. Biol. Chem. 270, 14024 –14030. 25. Yamano, Y., Ohyama, K., Chaki, S., Guo, D. F., and Inagami, T. (1992) Biochem. Biophys. Res. Commun. 187, 1426 –1431. 26. Hunyady, L., Bor, M., Balla, T., and Catt K, J. (1995) J. Biol. Chem. 270, 9702–9705. 27. Monnot, C., Bihoreau, C., Conchon, S., Curnow, K. M., Corvol, P., and Clauser, E. (1996) J. Biol. Chem. 271, 1507–1513. 28. Bihoreau, C., Monnot, C., Davies, E., Teutsch, B., Bernstein, K. E., and Corvol, P. (1993) Proc. Natl. Acad. Sci.USA 90, 5133– 5137. 29. Lokuta, A. J., Cooper, C., Gaa, S. T., Wang, H. E., and Rogers, T. B. (1994) J. Biol. Chem. 269, 4832– 4838. 30. Matute, C., Pulakat, L., Rio, C., Valcarcel, C., and Miledi, R. (1994) Proc. Natl. Acad. Sci. USA 91, 3774 –3778. 31. Smith, R. D., Corps, A. N., Hadfield, K. M., Vaughan, T. J., and Brown, K. D. (1994) Biochem. J. 302, 791– 800. 32. Yamada, T., Horiuchi, M., and Dzau, V. J. (1996) Proc. Natl. Acad. Sci. USA 93, 156 –160. 33. Stoll, M., Steckelings, U. M., Paul, M., Bottari, S. P., Metzger, R., and Unger, T. (1995) J. Clin. Invest. 95, 651– 657. 34. Pulakat, L., Tadessee, A. S., Dittus, J. J., and Gavini N. (1998) Reg. Peptides 73, 51–57. 35. Servant, G., Laporte, S. A., Leduc, R., Escher, E., and Guillemette, G. (1997) J. Biol. Chem. 272, 8653– 8659.
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Role of the His273 Located in the Sixth Transmembrane Domain of the Angiotensin II Receptor Subtype AT2 in Ligand–Receptor Interaction Cortney A. Turner, Shannon Cooper, and Lakshmidevi Pulakat 1 Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403
Received December 21, 1998
Angiotensin II receptor subtypes AT1 and AT2 are proteins with seven transmembrane domain (TMD) topology and share 34% homology. It was shown that His256, located in the sixth TMD of the AT1 receptor, is needed for the agonist activation by the Phe8 side chain of angiotensin II, although replacing this residue with arginine or glutamine did not significantly alter the affinity binding of the receptor. We hypothesized that the His273 located in the sixth transmembrane domain of the AT2 receptor may play a similar role in the functions of the AT2 receptor, although this residue was not identified as a conserved residue in the initial homology comparisions. Therefore, we replaced His273 of the AT2 receptor with arginine or glutamine and analyzed the ligand-binding properties of the mutant receptors using Xenopus oocytes as an expression system. Our results suggested that the AT2 receptor mutants His273Arg and His273 Glu have lost their affinity to [ 125I-Sar 1-Ile 8]Ang II, a peptidic ligand that binds both the AT1 and AT2 receptors and to 125 I-CGP42112A, a peptidic ligand that binds specifically to the AT2 receptor. Thus, His273 located in the sixth TMD of the AT2 receptor seems to play an important role in determining the binding properties of this receptor. Moreover, these results along with our previous observation that the Lys215 located in the 5th TMD of the AT2 receptor is essential for its high affinity binding to [ 125I-Sar 1-Ile 8]Ang II indicate that key amino acids located in the 5th and 6th TMDs of the AT2 receptor are needed for high affinity binding of the AT2 to its ligands. © 1999 Academic Press
Angiotensin II (Ang II) is an octapeptide hormone that is well known for its role in the regulation of cardiovascular functions and body fluid homeostasis [1–5]. Two major classes of cell surface receptors for Ang II, AT1 and AT2, have been cloned to date [6 –15]. 1
To whom correspondence should be addressed. Fax: (419) 372 2024. E-mail: [email protected]. 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
These receptors have different phamacological properties; the receptor subtype AT1 specifically binds a nonpeptidic ligand losartan (DUP753) and the receptor subtype AT2 specifically binds the peptidic ligand CGP42112A as well as the non-peptide ligand PD123319 [16 –20]. The AT1 receptor is responsible for mediating most of the known physiological effects of Ang II including the vasoconstriction, secretion of aldosterone and antidiuretic hormone and induction of thirst. This receptor is a peptide of 359 amono acids with seven transmembrane domains and belongs to the G-protein coupled-receptor family. The AT1 receptor is shown to activate both Gi and Gq type G-proteins. The AT1 receptor is also known to activate Jak/STAT proteins and mitogen activated protein kinases, and to induce cell proliferation [3, 20]. Detailed mutagenesis and molecular modeling studies have identified the key amino acids of the AT1 receptor involved in ligand binding and G-protein coupling [22–28]. The AT2 receptor is a peptide of 363 amino acids and shares 34% homology with the AT1 receptor. Like the AT1 receptor, the AT2 receptor also has a seven transmembrane domain topology, suggesting that this receptor also belongs to the G-protein coupled-receptor family. However, unlike the AT1 receptor, AT2 does not demonstrate the GTPgS-induced shift to a low affinity form that is characteristic of the G-protein-linked receptors. The AT2 receptor is known to act in a synergistic manner with the AT1 receptor in the activation of phospholipase A 2 and production of arachidonic acid in cardiac ischemia [29]. In contrast, the AT2 receptor is also known to exert opposing effects to that of the AT1 receptor on different cell types, since the AT2 receptor is shown to induce apoptosis whereas the AT1 receptor promotes cell proliferation [30 –32]. Both the AT1 and the AT2 receptor subtypes are often co-expressed [33]. Since these receptors may function in a synergistic manner in certain physiological conditions and in opposing manner in other physiological conditions, it is important to understand the
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FIG. 1. Predicted organization of rat AT2 receptor. Five putative N-linked glycosylation sites are marked (Y). The Histidine residue at position 273 in the sixth transmembrane domain of AT2 receptor that was subjected to mutagenesis is marked by the arrow.
similarities and differences between the structurefunction relationship of these receptors. The amino acid His256 is located in the sixth transmembrane domain of the AT1 receptor. Interaction between the Phe8 of Ang II and this His256 residue is shown to be essential for the agonist activation of the AT1 receptor [22]. The amino acid His273 is located in the sixth transmembrane domain of the AT2 receptor [14, 15]. Although initial comparisons of amino acid sequences of the AT1 and the AT2 receptors did not recognize this Histidine residue as a conserved residue [15], we felt that the location of this residue in the sixth transmembrane domain implies that this residue may play an analogus role to that of His256 of the AT1 receptor regarding the structure-function relationship of the AT2 receptor. Here we report the ligand-binding properties of two mutant AT2 receptors in which the His273 was replaced by glutamine or arginine. MATERIALS AND METHODS Materials. Restriction enzymes were purchased either from Boehringer Mannheim (Indianapolis, IN) or from Promega (Madison, WI). Oligonucleotides used for sequencing and mutagenesis were purchased from GIBCO BRL Life Technologies Inc. (Gaithersbureg, MD). Radiolabeled material for sequencing and binding studies ([ 35S]-dATP, [ 125I-Sar 1-Ile 8]Ang II, 125I-CGP42112A) were obtained from Dupont NEN (Boston, MA). Sequenase Version 2.0 DNA Sequencing Kit was from United States Biochemical, Cleveland, OH
and the DTth DNA Polymerase Sequencing Kit was from ClonTech, Palo Alto, CA. Mutagenesis was performed using Quick Change site-directed mutagenesis kit from Stratagene Products, La Jolla, CA. Riboprobe Gemini in vitro transcription system from Promega, Madison, WI was employed for in vitro transcription of the wild type and mutated AT2 receptors. Xenopus laevis were obtained from Xenopus One (Ann Arbor, MI). Mutagenesis of rat AT2 receptor. The rat AT2 receptor gene cloned in the pSP64 polyA vector [34] was used for the mutagenesis of the His273 codon using the Quick Change site-directed mutagenesis kit. Two synthetic oligonucleotides containing the desired mutation were used as primers [His273 Sense 27mer: 59-TGGCTTCCCTTCC(G/A)GGTTCTGACCTTC-39, His273 Antisense 27mer: 59-GAAGGTCAGAACC(T/C)GGAAGGGAAGCCA-39]. This was because double-stranded DNA was used as the template in this method. The mutated DNA was synthesized by Pfu DNA polymerase using the following thermocycling steps: Denaturing at 95°C for 2 minutes, Annealing at 55°C for 1 minute and then synthesizing at 68°C for 10 minutes. The program was repeated for 17 cycles and then the parental DNA template was digested using DpnI endonuclease which is specific for methylated and hemimethylated DNA. This DNA was then used for transforming E. coli TG1 cells and the mutants were identified by dideoxy nucleotide sequencing using DTth DNA Polymerase Sequencing Kit. In vitro transcription, expression in Xenopus oocytes and binding studies. In vitro transcription of the pSP64 poly(A) vectors carrying the wild type and mutant AT2 receptor genes was carried out using ‘Riboprobe Gemini Systems’ to generate cRNAs corresponding to the wild type and mutant AT2 receptor genes. Techniques for injection of cRNA were performed as described previously [34]. Oocytes were microinjected with 50 nl of cRNA solution at a concentration of 1 mg/ml and after two days the microinjected oocytes were treated
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FIG. 2. Ligand binding properties of the His273Arg and His273Gln mutants. The specific binding of the [ 125I-Sar 1-Ile 8]Ang II (0.5 nM) and I-CGP42112A (0.25 nM) to the oocytes expressing wild-type and mutated receptors are compared. Binding experiments were conducted for a period of one hour according to the procedures described previously [34]. Results are shown as relative % of the binding of the appropriate ligand to the oocytes expressing wildtype AT2 receptor. Receptor expression was quantitated by binding studies using 4 to 7 oocytes from at least 3 donors (a total of at least 12 oocytes). Three different cRNA preparations were used for each sample. Standard errors were with in 3% of each value. 125
with collagenase (0.5 mg/ml) for 20 minutes. The oocytes from each donor frog were screened to ensure that they did not express native Ang II receptors before injecting with AT2 receptor cRNAs by performing binding experiments with [ 125I-Sar 1-Ile 8]Ang II. The binding of [ 125I-Sar 1-Ile 8]Ang II or 125I-CGP42112A to single oocytes was carried out using the procedures described previously [34].
RESULTS AND DISCUSSION Mutagenesis and molecular modeling studies of the AT1 receptor have identified several key residues essential for the functions of this receptor. It has been shown that the amino acids Lys199 (located in the fifth transmembrane domain (TMD) of the AT1 receptor) and His256 (located in the sixth TMD of the AT1 receptor) play important roles in the binding of Ang II and agonist activation of the receptor, respectively [22]. It was also demonstrated that these residues play their role in the binding and activation of the AT1 receptor by directly interacting with the side chain of the Phe8 residue of Ang II. Previous amino acid comparisons between the AT1 and the AT2 receptors identified Lys215 located in the 5th TMD of this receptor as comparable to the Lys199 of the AT1 receptor [15]. Our mutational analysis showed that the Lys215 was essential for the binding of the [ 125I-Sar 1-Ile 8]Ang II to the AT2 receptor [34]. However, replacing this residue with Glutamic acid or Glutamine did not affect the afiinity of the receptor to the AT2 receptor specific peptidic ligand CGP42112A or the non-peptidic ligand PD123319. Thus, it seems that [ 125I-Sar 1-Ile 8]Ang II binding to rat AT2 receptor is comparable to the bind-
ing of this ligand to the rat AT1A receptor. However, binding requirements of the AT2 receptor-specific peptidic ligand, CGP42112A, are not similar to that of [ 125I-Sar 1-Ile 8]Ang II, or other peptidic ligands of AT1 receptor, since replacing Lys215 with Gln or Glu did not impair the affinity of this receptor to CGP42112A. Since His256 located in the sixth TMD was the other key amino acid of the AT1 receptor that directly interacts with the Phe8 of Ang II, and caused agonist activation of the receptor, we analyzed to see if the sixth TMD of the AT2 receptor also contained a Histidine residue that may function in a similar manner to that of His256. We identified His273 located in the sixth transmembrane domain of the AT2 receptor as the putative candidate (Figure 1), although this residue was not identified as a conserved residue in the previous homology comparisons (15). Since replacing the His256 with Arg or Gln did not affect the ligand binding properties of the AT1 receptor considerably [22], we analyzed to see if replacing His 273 by Arg or Gln influenced the ligand-binding properties of the AT2 receptor. To determine the ligand-binding properties of the mutated AT2 receptors, we microinjected Xenopus oocytes with equal amounts of cRNAs encoding the wildtype or the mutated AT2 receptors as described in the Materials and Methods. The follicular layer of the oocytes was removed by collagenase treatment followed by manual removal as described previously [34]. Ligand binding experiments were performed using [ 125ISar 1-Ile 8]Ang II, a peptidic ligand that binds both
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the AT1 and the AT2 receptor subtypes and 125ICGP42112A, a peptidic ligand that binds the AT2 receptor specifically. It was observed that both His273Arg and His273Gln mutants of the AT2 receptor have lost their affinity to [ 125I-Sar 1-Ile 8]Ang II and 125ICGP42112A (Figure 2). Thus His273 located in the sixth TMD of the AT2 receptor seems to be essential for the ability of the receptor to bind its peptidic ligands. Studies using photolabile Ang II analogs have shown that the amino terminal of the AT2 receptor interacts with the aminoterminal end of the Ang II and the the 3rd TMD of the AT2 receptor interacts with the carboxyl-terminal end of the Ang II [35]. Our mutagenesis studies suggest that as in the AT1 receptor, the amino acids located in the region spanning 5th and 6th TMD of the AT2 receptor also play important roles in the binding affinity of the AT2 receptor. Moreover, our results also show that, replacing His273 of the AT2 receptor, a residue analogous to His256, with Arg or Gln, resulted in generating mutants that have lost affinity to two peptidic ligands [ 125I-Sar 1-Ile 8]Ang II and 125I-CGP42112A. Based on these observations we conclude, that the His273 of the AT2 receptor is directly involved in determining the ligand-binding properties of the AT2 receptor. ACKNOWLEDGMENTS We also thank the members of Gavini/Pulakat laboratories at BGSU for their technical help during the course of this work and for helpful discussions. We thank B. Randall and D. Pax for expert animal care. This work is supported by NIH, National Heart, Lung, and Blood Institute Grant, HL60241 to L.P. This work is also supported by Research Challenge Grant, Faculty Research Committee Grant and Faculty Graduate Research Assistantship from BGSU to L.P.
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