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Role of Arg182 in the Second Extracellular Loop of Angiotensin II Receptor AT2 in Ligand Binding
Biochemical and Biophysical Research Communications 263, 816 – 819 (1999) Article ID bbrc.1999.1405, available online at http://www.idealibrary.com on
Role of Arg182 in the Second Extracellular Loop of Angiotensin II Receptor AT2 in Ligand Binding Jayson Kurfis, Dieter Knowle, and Lakshmidevi Pulakat 1 Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403
Received August 12, 1999
The phenolic side chain of Tyr 4 present in Ang II is proposed to interact with the side chain of Arg 167 of the AT1 receptor. To determine the contribution of the analogous Arg182 in the ligand-binding properties of the AT2, we replaced the Arg182 with Glu and Ala, and analyzed the ligand-binding properties. Our results suggest that replacing Arg182 with either Glu or Ala abolished the ability of the AT2 receptor to bind the nonspecific peptidic ligands, 125I-Ang II and [ 125I-Sar 1Ile 8]Ang II, as well as the AT2 receptor-specific peptidic ligand 125I-CGP42112A. We have shown previously that replacing the positively charged side chain of Lys215 with the negatively charged side chain of Glu in the fifth TMD did not alter the high affinity binding of 125I-CGP42112A to the AT2 receptor. However, ligand-binding properties of the Arg182Glu mutant suggest that positively charged side chain of Arg182 located in the junction of second ECL and the fourth TMD is critical for high affinity binding of all three peptidic ligands to the AT2 receptor. © 1999 Academic Press
The multifunctional peptide hormone, Angiotensin II (Ang II), is well known for its important roles in the regulation of cardiovascular and bodyfluid homeostasis (1–5). High affinity receptors implicated in mediating the effects of Ang II have been identified in a number of peripheral tissues such as those of the heart, mesenteric artery, aorta, adrenal cortex, liver, uterus, bladder and pituitary as well as in the brain (6 –12). To date two different Ang II receptor subtypes that demonstrate different pharmacological properties have been cloned. The Ang II receptor subtype having a high affinity for losartan, a non-peptide antagonist, has been designated AT1 (6 –10). This receptor is a 359amino acid long protein and has two subtypes, AT1A Abbreviations used: Ang II, Angiotensin II; GTPgS, guanosine 59-3-O-(thio)triphosphate; PCR, polymerase chain reaction; AT1 receptor, type 1 angiotensin II receptor; AT2 receptor, type II angiotensin II receptor. 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.
and AT1B. This receptor seems to mediate most of the well-known physiological effects of the Ang II such as vasoconstriction, aldosterone release and the dipsogenic effect and is known to couple to a variety of intracellular protiens such as G i, G q, Jak/STAT proteins and mitogen activated proteins (12–16). The other receptor subtype with high affinity for PD123319, a non-peptide antagonist and CGP42112A, a peptidic ligand has been designated AT2 (17–20). The AT2 receptor is a 363-amino acid long protein that shares only 34% homology with the AT1 receptor. Similar to the AT1 receptor, the AT2 receptor is a protein with seven transmembrane domain topology and shares 34% homology with the AT1 receptor (17–20). Activation of this receptor is shown to induce inhibition of cell proliferation and induction of apoptosis in many cell lines (21, 22). Mutational analysis and molecular modelling has provided a wealth of information regarding how Ang II binds to the AT1 receptor (22–26). It was proposed that the residues Tyr 4, His 6 and Phe 8 of Ang II are essential for its interaction with the AT1 receptors. The positively charged side arm of Lys199 located in the 5th transmembrane domain (TMD) of the AT1 receptor is reported to interact with the C-terminal carboxylate of Ang II and other peptidic ligands (22, 23). However, our previous studies have shown that, the AT2 receptor-specific, peptidic ligand CGP42112A could bind a mutant AT2 receptor in which the Lys215 (analogous to Ly199 of the AT1 receptor) was replaced by Glutamic acid (negatively charged side arm) with high affinity (27). In contrast, [Sar 1-Ile 8]Ang II, a peptidic ligand that binds both the AT1 and the AT2 receptors lost its affinity to the Lys215Glu mutant (27). Thus, the AT2 receptor seemed to have different requirements for the high affinity binding of these two peptidic ligands. The Arg167 of the AT1 receptor located at the junction of second extracellular loop and fourth TMD is shown to be essential for the ligand-binding of this receptor and it was suggested that the side chain of this Arginine extends towards the phenolic side chain of Tyr 4 residue present in Ang II (23, 26). The residue
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS method. The following thermocycling steps were used to synthesize the mutated DNA by Pfu DNA polymerase: 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. To generate cRNAs corresponding to the wild type and mutant AT2 receptor genes, 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’ for SP 6 RNA polymerase. Techniques for microinjection of cRNA were performed as described previously (27). The binding of [ 125I-Sar 1-Ile 8]Ang II, 125 I-Ang II or 125I-CGP42112A to single oocytes was carried out using the procedures described previously (27).
FIG. 1. Amino acid sequence and predicted organisation of the fourth and fifth transmembrane domains and second extracellular loop of the AT2 receptor. The Arg182 located at the junction of the fourth transmembrane domain and the second extracellular loop is shown in dark gray. The Lys215 located in the fifth transmembrane domain is shown in light gray.
Arg182 of the AT2 receptor is located at the junction of the second extracellular loop and the fourth TMD (Fig. 1). Since CGP42112A (the AT2 receptor-specific peptidic ligand) as well as Ang II and [Sar 1-Ile 8]Ang II (the peptidic ligands that bind both the AT1 and AT2 receptors) contain Tyr 4, we analysed if the contribution of Arg182 is similar in determining the high-affinity binding to the AT2 receptor to these ligands. Here we report the ligand-binding properties of two mutated AT2 receptors in which the Arg182 was replaced by either Glutamic Acid or Alanine. MATERIALS AND METHODS Materials. Oligonucleotides used for mutagenesis and sequencing were purchased from GIBCO BRL Life Technologies Inc. (Gaithersburg, MD). Mutagenesis was performed using Quick Change sitedirected mutagenesis kit from Stratagene Products (La Jolla, CA). 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). Restriction enzymes were purchased either from Boehringer Mannheim (Indianapolis, IN) or from Promega (Madison, WI). 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. To generate mutants of AT2 receptor, initially, we cloned rat AT2 receptor from rat fetus cDNA as described previously (27). The mutants of AT2 receptor were generated by using ‘Quick Change’ site-directed PCR mutagenesis kit. The oligonucleotide primers used to make Arg182Ala and Arg182Glu mutations were 59-GGTTCTGACATCT(T/C)CGAAATAAAATGTTGG-39 and 59-CCAACATTTTATTTCG(C/A)CGATGTCAGAACC. This was because double-stranded DNA was used as the template in this
RESULTS AND DISCUSSION As mentioned above, the amino acids Phe 8, His 6 and Tyr 4 are shown to be essential for the binding of Ang II to the AT1 receptor. Mutagenesis studies have shown that Phe 8 of Ang II interacts with Lys199 and His256 of the AT1. The docking of Ang II and other peptidic ligands to the AT1 receptor require interaction between the C-terminal carboxylate (Phe 8 or Ile 8) of the peptidic ligands and the positively charged sidearm of the Lys199. Agonist activation of the AT1 receptor requires an interaction between the His256 and the Phe 8 of Ang II. However, His256 is not essential for the high affinity binding of the ligand. Our previous studies have shown that the AT1 and AT2 receptors have slightly different requirements for their high affinity binding. For example, replacing the positively charged Lys215 (analogous to Lys199 or the AT1 receptor) with the negatively charged glutamic acid did not affect the affinity of the AT2 receptor to CGP42112A, the AT2 receptor specific ligand, whereas it abolished the affinity of the receptor to [Sar 1-Ile 8]Ang II, a ligand that can bind both Ang II receptors (27). This result suggested that the high affinity binding form of the AT2 receptor to CGP42112A is different from the high affinity binding form of the AT2 receptor to [Sar 1-Ile 8]Ang II, although both ligands have isoleucine as the C-terminal residue. Moreover, we have also shown that, unlike His256 of the AT1 receptor, the amino acid His273 (analogous to His256 of the AT1 receptor) located in the sixth transmembrane domain of the AT2 receptor is essential for the high affinity binding of the AT2 to both CGP42112A and [Sar 1Ile 8]Ang II (28). Replacing His272 with glutamine or arginine abolished the ability of the AT2 receptor to bind either CGP42112A or [Sar 1-Ile 8]Ang II. These results suggest that the contribution of different conserved amino acids to the high affinity binding of the AT1 and the AT2 receptors may differ. The amino acid Arg167 located at the junction of the second extracel-
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FIG. 2. Ligand binding properties of the Xenopus oocytes expressing Arg182Glu and Arg182Ala mutants of the AT2 receptor. The specific binding of the [ 125I-Sar 1-Ile 8]Ang II, 125I-Ang II and 125 I-CGP42112A (ligands at a concentration of 0.5 nM) to the oocytes expressing wildtype and mutated receptors are compared. Binding experiments were conducted for a period of one hour according to the procedures described previously (27). 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 6 oocytes from at least 5 donors (a total of at least 20 oocytes). Five different cRNA preparations were used for each sample.
lular loop (ECL) and fourth TMD of the AT1 receptor is shown to be crucial for its high affinity binding to both peptidic ligands and non-peptidic ligands and was suggested to be involved in interaction with Tyr 4, another key residue of Ang II (23, 26). Since Arg182 of the AT2 receptor is also located at the junction of the fourth TMD and the second ECL it was interesting to analyze how Arg182Glu and Arg182Ala mutants differ from wildtype in their high affinity binding requirements. The Xenopus oocytes were microinjected 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 follwed by manual removal as described previously (27). Ligand-binding experiments were performed using 125I-Ang II, a peptidic agonist that binds both AT1 and AT2 receptors, [ 125I-Sar 1-Ile 8]Ang II, a peptidic antagonist that binds both the AT1 and the AT2 receptor subtypes and 125ICGP42112A, a peptidic ligand that binds the AT2 receptor specifically. To determine the specific binding of each ligand, the ligand-binding experiments were carried out either in the presence of or in the absence of the AT2 receptor specific antagonist, PD123319 at a concentration of 1 mM. It was observed that both Arg182Glu and Arg182Ala mutants of the AT2 receptor have lost their affinity to [ 125I-Sar 1-Ile 8]Ang II and 125 I-Ang II, since no detectable specific binding was observed in either case (Fig. 2). Both mutants also had significant loss of affinity to 125I-CGP42112A (Fig. 2). Thus Arg182 located in the second extracellular loop seem to be essential for the high affinity binding of the
AT2 receptor to all three ligands used in this study. Since [ 125I-Sar 1-Ile 8]Ang II and 125I-Ang II are ligands that bind both the AT1 and the AT2, it was not surprising to see that Arg182Glu and Arg182Ala mutants were unable to bind these ligands with high affinity. This result would imply that the Arg182 of the AT2 receptor play a similar role to that of the AT1 receptor in determining the high affinity binding form of the receptor to its peptidic ligands. However, the observation that Arg182Glu and Arg182Ala mutants also lost affinity to the AT2 receptor specific peptidic ligand CGP42112A suggested that unlike Lys215, the contribution of Arg182 in determining the high affininty binding form of the receptor to the ligands that bind both the AT1 and the AT2 ([ 125I-Sar 1-Ile 8]Ang II and 125 I-Ang II) and that bind the AT2 specifically ( 125ICGP42112A) could be similar. ACKNOWLEDGMENTS 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. We 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.
REFERENCES 1. Dzau, V. J., Mukoyama, M., and Pratt, R. E. (1994) J. Hypertension Suppl. 12, S1–S5. 2. Lifton, R. P. (1995) Proc. Natl. Acad. Sci. USA 92, 8545– 8551. 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. 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. 5. de Gasparo, M., Rogg, H., Brink, M., Wang, L., Whitebread, S., Bullock, G., and Erne, P. (1994) Eur. Heart Journal. 15, Suppl D, 98 –103. 6. Chang, R. S. L., and Lotti, V. J. (1991). Life Sci. 49, 1485–1490. 7. 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. 8. 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. 9. Castern, E., and Saavedra, J. M. (1989) Proc. Natl. Acad. Sci. USA 86, 725–729. 10. Allen, A. M., Zhuo, J., and Mendelsohn, F. (1999) J. Am. Soc. Nephrol. 10, S23–S29 11. Matute, C., Pulakat, L., Rio, C., Valcarcel, C., and Miledi, R. (1994) Proc. Natl. Acad. Sci. USA 91, 3774 –3778. 12. Murphy, T. J., Alexander, R. W., Griendling, K., Runge, M. S., and Bernstein, K. E. (1991) Nature 351, 233–236. 13. Sassaki, K., Yamono, Y., Bardhan, S., Iwai, N., Murray, J. J.,
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Hasegawa, M., Matsuda, Y. and Inagami, T. (1991) Nature 351, 230 –232. Sandberg, K., Ji, H., Clark, J. A. L., Shapira, H., and Catt, K. J. (1992) J. Biol. Chem. 267, 9455–9458. Iwai, N., and Inagami, T. (1992) FEBS Lett. 298, 257–260. Marrero, M. B., Schieffer, B., Paxton, W. G., Heerdt, L., Berk, B. C., Delafontaine, P., and Bernstein, K. E. (1955). Nature 375, 247–250. Kambayashi, Y., Bardhan, S., Takahashi, K., Tsuzuki, S., Inui, H., Hamakubo, T., and Inagami, T. (1993) J. Biol. Chem. 268, 24543–24546. Mukoyama, M., Nakajima, M., Horiuchi, M., Sasamura, H., Pratt, R. E., and Dzau, V. J. (1993) J. Biol. Chem. 268, 24539 –24542. Tsuzuki, S., Ichiki, T., Nakakubo, H., Kitami, Y., Guo, D. F., Shirai, H., and Inagami, T. (1994) Biochem. Biophys. Res. Commun. 200, 1449 –1454. Yamada, T., Horiuchi, M., and Dzau, V. J. (1996) Proc. Natl. Acad. Sci. USA 93, 156 –160.
21. Stoll, M., Steckelings, U. M., Paul, M., Bottari, S. P., Metzger, R., and Unger, T. (1995) J. Clin. Invest. 95, 651– 657. 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. Feng, Y-H., Noda, K., Saad, Y., Liu, X., Husain, A., and Karnik, S. S. (1995) J. Biol. Chem. 270, 12846 –12850. 25. 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. 26. Yamano, Y., Ohyama, K., Chaki, S., Guo, D. F., and Inagami, T. (1992) Biochem. Biophys. Res. Commun. 187, 1426 –1431. 27. Pulakat, L., Tadessee, A. S., Dittus, J. J., and Gavini, N. (1998) Reg. Peptides 73, 51–57. 28. Turner, C. A., Cooper, S., and Pulakat, L (1999) Biochem. Biophys. Res. Commun. 257, 704 –707.
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Role of Arg182 in the Second Extracellular Loop of Angiotensin II Receptor AT2 in Ligand Binding Jayson Kurfis, Dieter Knowle, and Lakshmidevi Pulakat 1 Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403
Received August 12, 1999
The phenolic side chain of Tyr 4 present in Ang II is proposed to interact with the side chain of Arg 167 of the AT1 receptor. To determine the contribution of the analogous Arg182 in the ligand-binding properties of the AT2, we replaced the Arg182 with Glu and Ala, and analyzed the ligand-binding properties. Our results suggest that replacing Arg182 with either Glu or Ala abolished the ability of the AT2 receptor to bind the nonspecific peptidic ligands, 125I-Ang II and [ 125I-Sar 1Ile 8]Ang II, as well as the AT2 receptor-specific peptidic ligand 125I-CGP42112A. We have shown previously that replacing the positively charged side chain of Lys215 with the negatively charged side chain of Glu in the fifth TMD did not alter the high affinity binding of 125I-CGP42112A to the AT2 receptor. However, ligand-binding properties of the Arg182Glu mutant suggest that positively charged side chain of Arg182 located in the junction of second ECL and the fourth TMD is critical for high affinity binding of all three peptidic ligands to the AT2 receptor. © 1999 Academic Press
The multifunctional peptide hormone, Angiotensin II (Ang II), is well known for its important roles in the regulation of cardiovascular and bodyfluid homeostasis (1–5). High affinity receptors implicated in mediating the effects of Ang II have been identified in a number of peripheral tissues such as those of the heart, mesenteric artery, aorta, adrenal cortex, liver, uterus, bladder and pituitary as well as in the brain (6 –12). To date two different Ang II receptor subtypes that demonstrate different pharmacological properties have been cloned. The Ang II receptor subtype having a high affinity for losartan, a non-peptide antagonist, has been designated AT1 (6 –10). This receptor is a 359amino acid long protein and has two subtypes, AT1A Abbreviations used: Ang II, Angiotensin II; GTPgS, guanosine 59-3-O-(thio)triphosphate; PCR, polymerase chain reaction; AT1 receptor, type 1 angiotensin II receptor; AT2 receptor, type II angiotensin II receptor. 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.
and AT1B. This receptor seems to mediate most of the well-known physiological effects of the Ang II such as vasoconstriction, aldosterone release and the dipsogenic effect and is known to couple to a variety of intracellular protiens such as G i, G q, Jak/STAT proteins and mitogen activated proteins (12–16). The other receptor subtype with high affinity for PD123319, a non-peptide antagonist and CGP42112A, a peptidic ligand has been designated AT2 (17–20). The AT2 receptor is a 363-amino acid long protein that shares only 34% homology with the AT1 receptor. Similar to the AT1 receptor, the AT2 receptor is a protein with seven transmembrane domain topology and shares 34% homology with the AT1 receptor (17–20). Activation of this receptor is shown to induce inhibition of cell proliferation and induction of apoptosis in many cell lines (21, 22). Mutational analysis and molecular modelling has provided a wealth of information regarding how Ang II binds to the AT1 receptor (22–26). It was proposed that the residues Tyr 4, His 6 and Phe 8 of Ang II are essential for its interaction with the AT1 receptors. The positively charged side arm of Lys199 located in the 5th transmembrane domain (TMD) of the AT1 receptor is reported to interact with the C-terminal carboxylate of Ang II and other peptidic ligands (22, 23). However, our previous studies have shown that, the AT2 receptor-specific, peptidic ligand CGP42112A could bind a mutant AT2 receptor in which the Lys215 (analogous to Ly199 of the AT1 receptor) was replaced by Glutamic acid (negatively charged side arm) with high affinity (27). In contrast, [Sar 1-Ile 8]Ang II, a peptidic ligand that binds both the AT1 and the AT2 receptors lost its affinity to the Lys215Glu mutant (27). Thus, the AT2 receptor seemed to have different requirements for the high affinity binding of these two peptidic ligands. The Arg167 of the AT1 receptor located at the junction of second extracellular loop and fourth TMD is shown to be essential for the ligand-binding of this receptor and it was suggested that the side chain of this Arginine extends towards the phenolic side chain of Tyr 4 residue present in Ang II (23, 26). The residue
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS method. The following thermocycling steps were used to synthesize the mutated DNA by Pfu DNA polymerase: 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. To generate cRNAs corresponding to the wild type and mutant AT2 receptor genes, 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’ for SP 6 RNA polymerase. Techniques for microinjection of cRNA were performed as described previously (27). The binding of [ 125I-Sar 1-Ile 8]Ang II, 125 I-Ang II or 125I-CGP42112A to single oocytes was carried out using the procedures described previously (27).
FIG. 1. Amino acid sequence and predicted organisation of the fourth and fifth transmembrane domains and second extracellular loop of the AT2 receptor. The Arg182 located at the junction of the fourth transmembrane domain and the second extracellular loop is shown in dark gray. The Lys215 located in the fifth transmembrane domain is shown in light gray.
Arg182 of the AT2 receptor is located at the junction of the second extracellular loop and the fourth TMD (Fig. 1). Since CGP42112A (the AT2 receptor-specific peptidic ligand) as well as Ang II and [Sar 1-Ile 8]Ang II (the peptidic ligands that bind both the AT1 and AT2 receptors) contain Tyr 4, we analysed if the contribution of Arg182 is similar in determining the high-affinity binding to the AT2 receptor to these ligands. Here we report the ligand-binding properties of two mutated AT2 receptors in which the Arg182 was replaced by either Glutamic Acid or Alanine. MATERIALS AND METHODS Materials. Oligonucleotides used for mutagenesis and sequencing were purchased from GIBCO BRL Life Technologies Inc. (Gaithersburg, MD). Mutagenesis was performed using Quick Change sitedirected mutagenesis kit from Stratagene Products (La Jolla, CA). 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). Restriction enzymes were purchased either from Boehringer Mannheim (Indianapolis, IN) or from Promega (Madison, WI). 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. To generate mutants of AT2 receptor, initially, we cloned rat AT2 receptor from rat fetus cDNA as described previously (27). The mutants of AT2 receptor were generated by using ‘Quick Change’ site-directed PCR mutagenesis kit. The oligonucleotide primers used to make Arg182Ala and Arg182Glu mutations were 59-GGTTCTGACATCT(T/C)CGAAATAAAATGTTGG-39 and 59-CCAACATTTTATTTCG(C/A)CGATGTCAGAACC. This was because double-stranded DNA was used as the template in this
RESULTS AND DISCUSSION As mentioned above, the amino acids Phe 8, His 6 and Tyr 4 are shown to be essential for the binding of Ang II to the AT1 receptor. Mutagenesis studies have shown that Phe 8 of Ang II interacts with Lys199 and His256 of the AT1. The docking of Ang II and other peptidic ligands to the AT1 receptor require interaction between the C-terminal carboxylate (Phe 8 or Ile 8) of the peptidic ligands and the positively charged sidearm of the Lys199. Agonist activation of the AT1 receptor requires an interaction between the His256 and the Phe 8 of Ang II. However, His256 is not essential for the high affinity binding of the ligand. Our previous studies have shown that the AT1 and AT2 receptors have slightly different requirements for their high affinity binding. For example, replacing the positively charged Lys215 (analogous to Lys199 or the AT1 receptor) with the negatively charged glutamic acid did not affect the affinity of the AT2 receptor to CGP42112A, the AT2 receptor specific ligand, whereas it abolished the affinity of the receptor to [Sar 1-Ile 8]Ang II, a ligand that can bind both Ang II receptors (27). This result suggested that the high affinity binding form of the AT2 receptor to CGP42112A is different from the high affinity binding form of the AT2 receptor to [Sar 1-Ile 8]Ang II, although both ligands have isoleucine as the C-terminal residue. Moreover, we have also shown that, unlike His256 of the AT1 receptor, the amino acid His273 (analogous to His256 of the AT1 receptor) located in the sixth transmembrane domain of the AT2 receptor is essential for the high affinity binding of the AT2 to both CGP42112A and [Sar 1Ile 8]Ang II (28). Replacing His272 with glutamine or arginine abolished the ability of the AT2 receptor to bind either CGP42112A or [Sar 1-Ile 8]Ang II. These results suggest that the contribution of different conserved amino acids to the high affinity binding of the AT1 and the AT2 receptors may differ. The amino acid Arg167 located at the junction of the second extracel-
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FIG. 2. Ligand binding properties of the Xenopus oocytes expressing Arg182Glu and Arg182Ala mutants of the AT2 receptor. The specific binding of the [ 125I-Sar 1-Ile 8]Ang II, 125I-Ang II and 125 I-CGP42112A (ligands at a concentration of 0.5 nM) to the oocytes expressing wildtype and mutated receptors are compared. Binding experiments were conducted for a period of one hour according to the procedures described previously (27). 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 6 oocytes from at least 5 donors (a total of at least 20 oocytes). Five different cRNA preparations were used for each sample.
lular loop (ECL) and fourth TMD of the AT1 receptor is shown to be crucial for its high affinity binding to both peptidic ligands and non-peptidic ligands and was suggested to be involved in interaction with Tyr 4, another key residue of Ang II (23, 26). Since Arg182 of the AT2 receptor is also located at the junction of the fourth TMD and the second ECL it was interesting to analyze how Arg182Glu and Arg182Ala mutants differ from wildtype in their high affinity binding requirements. The Xenopus oocytes were microinjected 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 follwed by manual removal as described previously (27). Ligand-binding experiments were performed using 125I-Ang II, a peptidic agonist that binds both AT1 and AT2 receptors, [ 125I-Sar 1-Ile 8]Ang II, a peptidic antagonist that binds both the AT1 and the AT2 receptor subtypes and 125ICGP42112A, a peptidic ligand that binds the AT2 receptor specifically. To determine the specific binding of each ligand, the ligand-binding experiments were carried out either in the presence of or in the absence of the AT2 receptor specific antagonist, PD123319 at a concentration of 1 mM. It was observed that both Arg182Glu and Arg182Ala mutants of the AT2 receptor have lost their affinity to [ 125I-Sar 1-Ile 8]Ang II and 125 I-Ang II, since no detectable specific binding was observed in either case (Fig. 2). Both mutants also had significant loss of affinity to 125I-CGP42112A (Fig. 2). Thus Arg182 located in the second extracellular loop seem to be essential for the high affinity binding of the
AT2 receptor to all three ligands used in this study. Since [ 125I-Sar 1-Ile 8]Ang II and 125I-Ang II are ligands that bind both the AT1 and the AT2, it was not surprising to see that Arg182Glu and Arg182Ala mutants were unable to bind these ligands with high affinity. This result would imply that the Arg182 of the AT2 receptor play a similar role to that of the AT1 receptor in determining the high affinity binding form of the receptor to its peptidic ligands. However, the observation that Arg182Glu and Arg182Ala mutants also lost affinity to the AT2 receptor specific peptidic ligand CGP42112A suggested that unlike Lys215, the contribution of Arg182 in determining the high affininty binding form of the receptor to the ligands that bind both the AT1 and the AT2 ([ 125I-Sar 1-Ile 8]Ang II and 125 I-Ang II) and that bind the AT2 specifically ( 125ICGP42112A) could be similar. ACKNOWLEDGMENTS 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. We 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.
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