Introduction

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Angiotensin II (Ang II) is a circulating and local tissue hormone regulating fluid and electrolyte metabolism, hormone secretion, the autonomic nervous system, and brain function (Saavedra, 1992; Matsusaka and Ichikawa, 1997; Timmermans, 1999). There are two types of mammalian Ang II receptors, AT₁ and AT₂. AT₁ receptors are selectively antagonized by nonpeptidic biphenylamidazoles or by imidazoleacrylic compounds (Timmermans, 1999). Stimulation of AT₁ receptors modulates Ang II effects, and nonpeptide AT₁ receptor antagonists are used in hypertension treatment (Timmermans, 1999).

Ang II AT₁ receptors belong to the seven-transmembrane G-protein-coupled receptor superfamily (Sandberg, 1994). Mammalian AT₁ receptors have higher than 90% amino acid sequence identity and similar affinity for peptide ligands, such as Ang II. Many mammalian AT₁ receptors express an affinity for the biphenylamidazole antagonist losartan in the same range as human AT₁ receptors (Chiu et al., 1993), but losartan affinity is reduced in bovine, canine, ferret, and porcine AT₁ receptors (Chiu et al., 1993; Itazaki et al., 1993; Burns et al., 1994; Gosselin et al., 2000). Amphibian Ang II receptors, with lower homology to mammalian AT₁ receptors, also bind peptide ligands with similar affinity (Sandberg, 1994) but have affinity for losartan several orders of magnitude lower that that of mammalian receptors (Ji et al., 1995). This suggests that the nonpeptide binding domain was largely distinct from the receptor domain involved in Ang II binding (De Gasparo et al., 2000). Such differences between domains involved in the recognition of peptide and nonpeptide ligands hold true for many other G-protein-coupled receptors (Beinborn et al., 1993; Gether et al., 1993; Kong et al., 1994).

For AT₁ receptors, important epitopes involved in Ang II binding may be located around the top of transmembrane segments I, II, and VII, in close spatial proximity in the folded receptor structure (Hjorth et al., 1994). Binding of nonpeptide Ang II antagonists may be dependent on nonconserved residues located deep in the hydrophobic transmembrane segments of the AT₁ receptor, as demonstrated by mutational analysis of the mammalian AT₁ and amphibian Ang II receptors (Bihoreau et al., 1993; Ji et al., 1993, 1994, 1995; Marie et al., 1994; Schambye et al., 1994; Noda et al., 1995; Monnot et al., 1996; Inoue et al., 1997; De Gasparo et al., 2000).

In rats and mice, there are two AT₁ receptor subtypes (AT_1A and AT_1B) encoded by different genes and with significant sequence homology in the coding regions [open-reading frames (ORF)] (Iwai and Inagami, 1992; Sasamura et al., 1992; Yoshida et al., 1992). AT_1A and AT_1B receptors have similar affinity for nonpeptide receptor antagonists and cannot be differentiated pharmacologically, although they are differentially localized and regulated (Iwai and Inagami, 1992; Sasamura et al., 1992; Yoshida et al., 1992).

In another rodent species, the gerbil, we found an Ang II receptor subtype that had high affinity for Ang II but was unable to recognize nonpeptide antagonists (De Oliveira et al., 1995). Cloning the receptor from a gerbil kidney cDNA library revealed higher than 90% homology to mammalian AT₁ receptors and a difference from the hAT₁ receptor at only 25 amino acid residues (Moriuchi et al., 1998). The receptor expressed high affinity for Ang II, similar to the human AT₁ receptor, but greatly reduced (400-fold) affinity for losartan (Moriuchi et al., 1998). This gerbil receptor had a distribution similar to other rodent AT_1A receptors, with closer homology to rodent (rAT_1A and mAT_1A) receptors than to their AT_1B subtypes (Moriuchi et al., 1998). We considered this gerbil AT₁ receptor as a gAT_1A subtype (Moriuchi et al., 1998).

Gerbils transcribed an additional AT₁ receptor subtype that had affinity for Ang II similar to that of the hAT₁ receptor but an affinity for losartan intermediate between that of the hAT₁ and the gAT_1A receptor. This receptor was specifically localized to the adrenal zona glomerulosa, with an affinity for losartan 40-fold lower than that of the hAT₁ receptor (Moriuchi et al., 1998). We speculated that the second gerbil AT₁ receptor was in fact the rodent AT_1B subtype.

We initiated studies to identify the amino acid residues responsible for the reduced affinity to nonpeptide antagonists of the naturally mutated gAT₁ receptors. This presented a distinct advantage, because natural mutants probably keep the whole integrity of the three-dimensional structure for Ang II binding without major distortions. Site-directed mutagenesis of gerbil and human AT₁ receptors, with a combination of gain- and loss-of-function experiments, could further advance the identification of amino acids involved in the binding mechanism of nonpeptide antagonists.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Animals. We purchased male Mongolian gerbils (Meriones unguiculatus, 65-80 g) from Tumblebrook Farm (West Brookfield, MA). The animals were kept under standard conditions with food and water ad libitum and were killed by decapitation between 9:00 and 10:00 AM. To study receptor binding and in situ hybridization, adrenal glands and kidneys were dissected and frozen in isopentane on dry ice. Tissue sections (16-µm thickness) were cut in a cryostat and kept at 80°C until used. The National Institutes of Health Animal Care and Use Committee approved all animal procedures.

Materials. ¹²⁵I-Sar¹-Ang II (specific activity, 2200 Ci/mmol) and RNA labeling kits were purchased from PerkinElmer Life Sciences (Boston, MA). Ang II was purchased from Peninsula Laboratories (Belmont, CA). ³⁵S-UTP (specific activity, >1000 Ci/mmol) and Hyperfilm-³H were obtained from Amersham Biosciences (Piscataway, NJ). Losartan was a gift from DuPont Merck Pharmaceutical Co. (Wilmington, DE). Cell culture products (Opti-MEM and Dulbecco's modified Eagle's medium) and binding buffers [Hanks' balanced salt solution (HBSS)] were purchased from Invitrogen (Carlsbad, CA). The monkey kidney epithelial COS-7 cell line was obtained from American Type Culture Collection (Manassas, VA). Restriction enzymes were purchased from New England Biolabs (Beverly, MA). The cDNA synthesis kit, DNA labeling kit (Prime-It II), and QuikChange site-directed mutagenesis kit were obtained from Stratagene (La Jolla, CA). The pcDNA3.1 vector was purchased from Invitrogen. Nucleotide sequence analysis was performed using an ABI Prism 310 sequencing machine and a Big Dye Primer Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). The vector plasmid with the hAT₁ receptor cDNA insert was a generous gift from Dr. T. Inagami (Vanderbilt University School of Medicine, Nashville, TN).

Molecular Cloning of gAT_1B Receptor Gene. We constructed a cDNA library from gerbil adrenal gland mRNA and selected a cDNA insert in the range of about 1.8 to 4 kb. A complexity of 1 × 10⁶ independent clones was constructed in a  uni-ZAP vector. Independent clones were screened with a conventional in situ plaque hybridization method, using hAT₁ receptor ORF cDNA as a probe, and the positive clones were screened and hybridized again to a probe directed to the gAT_1A 3'-UTR. Selected clones containing the gAT_1B receptor gene were treated to make in vivo conversion from the  uni-ZAP vector into a phagemid vector, pBK-CMV, and were subject to further nucleotide sequence analysis using an ABI Prism 310 sequencing machine (Applied Biosystems).

In Situ Hybridization. A specific riboprobe directed to the 3'-UTR of the cloned gAT_1B receptor was obtained after subcloning of a polymerase chain reaction-generated DNA fragment of 729 bp into the XbaI-EcoRI site of the pBluescript II KS vector (Stratagene). The DNA fragment was amplified with XbaI-extended forward and EcoRI-containing reverse primers, corresponding to nucleotides 1128 through 1856. Antisense and sense (control) riboprobes were labeled by in vitro transcription in the presence of 200 µCi of ³⁵S-UTP (Amersham Biosciences; >1000 Ci/mmol). In situ hybridization was performed as described previously (Moriuchi et al., 1998), with modifications as follows: adrenal gland and kidney sections were covered with hybridization buffer containing 4 × 10⁴ cpm/µl of probe, hybridized for 18 h at 54°C, treated with RNase A, and washed with increasing stringency. After a final high-stringency wash in 0.1 × standard saline citrate at 65°C for 1 h, sections were dehydrated, exposed to Hyperfilm-³H for 4 days, and developed as described previously (Moriuchi et al., 1998).

Expression of Angiotensin II Receptors in COS-7 Cells. Monkey kidney epithelial COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 4.5 g/l of glucose, 4 mM glutamine, 10% fetal bovine serum, 100 units/ml of penicillin, and 100 µg/ml of streptomycin in a humidified atmosphere of 5% CO₂ and 95% air at 37°C. One day after plating in tissue culture plates (2 × 10⁶ cells/100 cm²), cells were washed with 10 ml of Opti-MEM. The gAT_1A, gAT_1B, and hAT₁ receptors and receptor chimera cDNAs were subcloned into the pcDNA3.1 vector. Four micrograms of vector DNA in 800 µl of Opti-MEM, and 16 µl of LipofectAMINE (Invitrogen) in 800 µl of Opti-MEM, were mixed and incubated at room temperature for 30 min. The DNA/LipofectAMINE complex was added to each plate after mixing with 6.4 ml of Opti-MEM. The next day, transfected cells were divided into wells of 24-well tissue culture plates (1 × 10⁵ cells/well).

Ligand Binding and Displacement Study. The ligand displacement analysis was performed the day after the split of the transfected cells into 24-well plates. Cells were washed with binding buffer (0.2% bovine serum albumin in HBSS) and incubated at room temperature for 2 h with 0.3 nM ¹²⁵I-Sar¹-Ang II with variable concentrations (10¹² to 10³ M) of the unlabeled competitive peptide agonist (Ang II) or the selective nonpeptidic AT₁ receptor antagonist losartan. After 2 h of incubation, the cells were washed with ice-cold HBSS three times, dissolved in 0.2 M NaOH, and bound ¹²⁵I-Sar¹-Ang II was measured in a gamma counter. Each experiment was carried out at least twice in triplicate. The binding data were analyzed, and EC₅₀ values were determined by computerized nonlinear regression analysis using GraphPad Prism 2.0 software (GraphPad Software, San Diego, CA). To compare relative affinities of multiple mutants, we compared them with the affinity of the wild-type hAT₁ receptor. We determined F_mut values as the ratio of mutant EC₅₀/hAT₁ EC₅₀ values.

Representation of the Presumed Binding Pocket for the Biphenylimidazole Antagonists. The sequence of the hAT₁ receptor was analyzed using the SOSUI system software (version 1.0) (Hirokawa et al., 1998) to obtain a two-dimensional representation (Schambye et al., 1994) and a helical wheel diagram (Murgolo et al., 1996). Positions considered important for losartan binding were identified based on the present results and those of the literature.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Molecular Cloning of the New Gerbil AT₁ Receptor. We constructed a cDNA library from gerbil adrenal gland, selected inserts in the range of 1.8 to 4 kb, and constructed a complexity of 1 × 10⁶ clones in a -uni-ZAP vector. We screened approximately 80,000 independent clones by conventional in situ plaque hybridization methods, using human AT₁ receptor ORF cDNA as a probe; we identified 19 positive clones. To exclude clones positive for gAT_1A, the positive clones were screened and hybridized again to a probe directed to the gAT_1A 3'-UTR. None of the 19 positive clones hybridized to this probe. Four different clones, designated as 3, 4, 6, and 9, were finally selected and thoroughly sequenced. The nucleotide sequence of the longest clone (clone 4; 2,745 bp), contained an ORF of 1,077 bp encoding a protein of 359 amino acid residues with a 3'-UTR 700 bp longer than that present in the other clones. Clone 9 had a 3'-UTR shorter than that of clone 4 but an extra 96-bp exon in 5'-UTR. Clones 3 and 6 had 3'-UTRs of similar length as that of clone 9, with the exception of the extra exon. The molecular characteristics of the cloned gerbil cDNA were similar to those of other mammalian AT₁ receptors (Moriuchi et al., 1998).

View larger version (70K): [in this window] [in a new window]   Fig. 1.   Alignment of the gerbil angiotensin II AT1B receptor amino acid sequence with those of other mammalian AT1 receptors. -represents identical amino acids. Amino acid differences are shown in their corresponding positions. Solid lines indicate prospective transmembrane domains I through VII. Amino acid positions are numbered on the right. The gerbil AT1B nucleotide sequence was deposited in GenBank (accession number AF 078794).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   A, autoradiograms of gerbil adrenal gland and kidney. Figures represent in situ hybridization with antisense or sense probes specific for the 3'-untranslated region of the cloned gerbil angiotensin II AT1B receptor cDNA. Note the positive signal in the gerbil adrenal zona glomerulosa and the absence of signal in the gerbil kidney with the antisense riboprobe. B, saturation isotherm of specific 125I-Sar1-Ang II binding to the gerbil angiotensin II AT1B receptor transfected to COS-7 cells. Insert shows a Scatchard plot. The figure represents a typical experiment repeated three times in triplicate.

Selection of Mutagenesis Sites. Our initial studies with gAT_1A and hAT₁ chimerical receptors identified the responsible region for the reduced affinity for losartan as located between the TM II and TM VII helices (amino acids 63-321) (R. Moriuchi and J. M. Saavedra, unpublished observations). Eight of the amino acids in the gAT_1A receptor that are different from the hAT₁ receptor were identified in this region (Val⁸³ in TM II; Gly¹⁰⁷ and Ile¹⁰⁸ in TM III; Val¹⁵⁰, Val¹⁵¹, and Val ¹⁶⁴ in TM IV; Ser¹⁷⁷ in the extracellular loop 2; and Met²⁰⁵ in TM V) replacing Leu⁸³, Ser¹⁰⁷, Val¹⁰⁸, Ile¹⁵⁰, Ile¹⁵¹, Ile¹⁶⁴ Ile¹⁷⁷, and Leu²⁰⁵ in hAT₁ receptors (Moriuchi et al., 1998).

Gain-of-Function Mutation in gAT_1A Receptors. We performed gain-of-function assays with gAT_1A mutants in single individual positions followed by the analysis of dual combinatorial mutants. Single-mutant analysis revealed substantial gain of function for mutants in both positions 107 and 108. The replacement of I108V was six times more effective than G107S (Table 1; Figure 3). The combinatorial replacement with G107S/I108V resulted in a binding affinity for losartan that was comparable with that of the wild-type hAT₁.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Gain-of-function mutations in gerbil AT1A receptor. The figure represents displacement of 125I-Sar1-Ang II binding, by increasing concentrations of losartan, for selected gAT1A mutant receptors transfected to COS-7 cells. The arrow shows the progressive gain of function compared with the hAT1 receptor. , hAT1; , gAT1A; , V83L; , G107S/I108V; , G107S; , I108V; , V150I/V151I; , S177I.

Combinatorial Gain-of-Function Mutations in Gerbil AT_1A. The combinatorial variant (G107S/I108V/V150I/V151I) obtained by addition of mutations V150I/V151I to the mutants G107S/I108V recovered its losartan-binding affinity to a value very similar to that of the hAT₁ wild-type receptor (F_mut = 1.1) (Table 2; Fig. 4), but the effect was not different from that of the mutants G107S/I108V alone (Table 1).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Gain-of-function multiple combinatorial mutations in gerbil AT1A receptor. The figure represents displacement of 125I-Sar1-Ang II binding, by increasing concentrations of losartan, for selected gAT1A mutant receptors transfected to COS-7 cells. The arrow shows the progressive gain of function compared with the hAT1 receptor. , hAT1; , gAT1A; , G107S/I108V/V150I; , G107S/I108V/S177I; , G107S/I108V/V150I/V151I/S177I; , G107S/I108V/V150I/V151I/S177I/V83L; , V150I/V151I/S177I; , V150I/V151I/S177I/V83L.

Gain-of-Function Mutation in gAT_1B Receptors. Our experiments revealed that single amino acid replacement (G107S or M195L) resulted in a 3- to 4-fold gain of function as evidenced by the increase in losartan-binding affinity (Table 3; Fig. 5A). The V151I mutant did not produce a detectable gain of function (Table 3; Fig. 5A). When the mutants G107S and/or M195L were combined, the gain of function increased to 13-fold, indicating that the contribution of these two sites is exclusive and additive (Table 3; Fig. 5B). However, the gain of function produced by the G107S/M195L mutant was incomplete, and the binding affinity for losartan was still 3-fold lower compared with the wild-type gAT_1B receptor (Table 3; Fig. 5B). As expected, the double mutant G107S/V151I exhibited a gain of function similar to that of the single G107S mutant (Table 3; Fig. 5, A and B). We obtained the following rank order for combinatorial mutants on losartan-binding affinity: hAT₁ < G107S/M195L < M195L = G107S/V151I = G107S < V151I = gAT_1B (Table 3).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   A, gain of function in gerbil AT1B receptor after single mutations. The figure represents displacement of 125I-Sar1-Ang II binding, by increasing concentrations of losartan, for selected gAT1B mutant receptors transfected to COS-7 cells. The arrow shows the progressive gain of function compared with the hAT1 receptor. , hAT1; , gAT1B; , G107S; , V151I; , M195L. B, gain of function in gerbil AT1B receptor after combinatorial mutations. The figure represents displacement of 125I-Sar1-Ang II binding, by increasing concentrations of losartan, for selected gAT1B mutant receptors transfected to COS-7 cells. The arrow shows the progressive gain of function compared with the hAT1 receptor. , hAT1; , gAT1B; , G107S/V151I; , G107S/M195L.

Loss-of-Function Mutation in gAT_1A and gAT_1B Receptors. To confirm the role of the amino acid in position 195, we constructed a L195M mutant on the gAT_1A receptor. The wild-type gAT_1A receptor exhibits a 400-fold lower binding affinity for losartan while retaining a conserved amino acid (Leu) in position 195. We found that the gAT_1A L195M mutant showed an additional 4-fold decrease in losartan binding (F_mut = 1389; Table 4; Fig. 6A).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   A, loss-of-function mutations in gerbil AT1A receptors. The figure represents displacement of 125I-Sar1-Ang II binding, by increasing concentrations of losartan, for selected gAT1A mutant receptors transfected to COS-7 cells. The arrow shows the progressive loss of function compared with the hAT1 receptor. , hAT1; , gAT1A; , gAT1B; , gAT1A+L195M. B, loss-of-function mutations in gerbil AT1B receptors. The figure represents displacement of 125I-Sar1-Ang II binding, by increasing concentrations of losartan, for selected gAT1B mutant receptors transfected to COS-7 cells. The arrow shows the progressive loss of function compared with the hAT1 receptor. , hAT1; , gAT1A; , gAT1B; , gAT1B+V108I.

Mutagenesis of hAT₁ Receptors with Loss-of-Function. To confirm the role of amino acids in positions 107, 108, and 195, we constructed hAT₁ mutants and determined the possibility of loss of function. The S107G mutant produced a 2.7-fold decrease in losartan-binding, whereas the V108I mutant decreased losartan-binding affinity 45-fold (Table 5; Fig. 8A). The double mutant S107G/V108I resulted in a more pronounced, 160-fold loss of function (Table 5; Fig. 7A).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   A. Loss-of-function mutations in human AT1 receptors (S107G and V108I). The figure represents displacement of 125I-Sar1-Ang II binding, by increasing concentrations of losartan, for selected hAT1 mutant receptors transfected to COS-7 cells. The arrow shows the progressive loss of function of the mutated hAT1 receptors. Single and combinatorial mutants are compared with the gAT1A receptor. , hAT1; , gAT1A; , gAT1B; , S107G; , V108I; , S107G/V108I. B, loss-of-function mutations in human AT1 receptors (L195M and combinatorial mutations). The figure represents displacement of 125I-Sar1-Ang II binding, by increasing concentrations of losartan, for selected hAT1 mutant receptors transfected to COS-7 cells. The arrow shows the progressive loss of function of the mutated hAT1 receptors. Single and combinatorial mutants are compared with the gAT1B receptor. , hAT1; , gAT1A; , gAT1B; , L195M; , S107G/L195M; , V108I/L195M; , V108I/S107G/L195M.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Mutagenesis at position Ile108 in the human AT1 and gerbil AT1A receptors. The figure represents displacement of 125I-Sar1-Ang II binding, by increasing concentrations of losartan, for selected mutant receptors transfected to COS-7 cells. The arrows show the effect of the different amino acid substitutions in the hAT1 and gAT1A receptors. , hAT1 Val108; , V108I; , gAT1A Ile108; , I108V; , I108A; , I108S.

Mutations at Position Ile¹⁰⁸. We compared the effects of different amino acids in position 108, the most important for losartan-binding affinity. The V108I mutant in the hAT₁ receptor decreased binding affinity 45-fold (Table 6; Fig. 8), whereas in the gAT_1A, with the natural mutant V108I, binding affinity was about 400-fold lower than in the hAT₁ receptor (Table 6; Fig. 8). In the gAT_1A receptor, the mutant I108V produced a significant gain of function and the mutant I108A resulted in a gain of function of a lesser degree (Table 6; Fig. 8). The Ala residue was better than the Ile residue but detrimental with respect to Val at position 108. Conversely, the mutant I108S in the gAT_1A receptor actually produced a significant loss of function compared with the wild type gAT_1A receptor (Table 6; Fig. 8). The rank order for loss-of-function mutations in position 108 was: hAT₁ < gAT_1A/I108V < hAT₁/V108I < gAT_1A I108A < gAT_1A < gAT_1A I108S (Table 6).

View larger version (35K): [in this window] [in a new window]   Fig. 9.   Two-dimensional representation of the human AT1 receptor obtained using the SOSUI sytem software. The representation is very similar to that described earlier for the human AT1 receptor (Schambye et al., 1994). Black circles denote the animo acid residues described herein as important for the binding of nonpeptidic antagonists. White triangles denote residues described by other authors. A white triangle inside a black circle denotes residues described both herein and in the literature.

View larger version (41K): [in this window] [in a new window]   Fig. 10.   Helical wheel diagram of the human AT1 receptor drawn using the SOSUI system software. The helices were oriented with the regions of highest hydrophobicity pointing to the exterior (i.e., lipid interface) of the helix bundle and charged residues mostly facing the center of the helix bundle (Murgolo et al., 1996). Squares denote the amino acid residues described herein as important for the binding of the nonpeptidic antagonist. Circles denote residues described in the literature. A circle inside a square denotes residues described both herein and in the literature.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

We report the cloning and characterization of a gerbil Ang II receptor, highly homologous to the hAT₁ and gAT_1A receptors, discreetly localized to the zona glomerulosa of the adrenal gland (present results and Moriuchi et al., 1998), with similar affinity to Ang II compared with the hAT₁ receptor and with low affinity for the nonpeptidic antagonist losartan, intermediate between the hAT₁ and the gAT_1A receptors (Moriuchi et al., 1998). Because of these characteristics, we considered the newly cloned receptor as a gAT_1B receptor.

We conclude based on our gain- and loss-of-function studies of the gAT_1A, gAT_1B, and hAT₁ receptors that: 1) the most important amino acid was Val¹⁰⁸, followed by Gly¹⁰⁷, and their role was cumulative; 2) an additional very important amino acid was Leu¹⁹⁵, but only in combination with Val¹⁰⁸ or Gly¹⁰⁷; and 3) we found additional but much lesser roles for Leu⁸³, Ile¹⁵⁰, Ile¹⁵¹, and Ile¹⁷⁷.

For losartan binding to AT₁ receptors, the most important amino acids are located in TM III. We found that Gly¹⁰⁷, previously considered not to be a major contributor to losartan binding (Ji et al., 1994), is important to losartan affinity for both the gerbil and hAT₁ receptors. The S107A mutation of rAT_1A did not affect losartan-binding affinity (Monnot et al., 1996). It is possible that only Gly¹⁰⁷ replacement has an effect on losartan binding, even though both Ala (R = CH3) and Gly (R = H) are nonpolar amino acids. This being the case, the presence of one additional carbon in the side chain in the R group could be critical for losartan binding, as is the case when Thr (2C) replacing Ser¹⁰⁹ (1C) reduces the binding affinity of rAT₁ receptor for losartan by 190-fold (Ji et al., 1995).

Using gain- and loss-of-function mutations, we confirm here that Val¹⁰⁸ is the most important amino acid for losartan binding to AT₁ receptors, both in gerbils and humans (Ji et al., 1995; Nirula et al., 1996; and present report). The gAT_1A receptor expresses both the I108V and the G107 mutations, and these are additive. On the other hand, Val¹⁰⁸ is conserved in the gAT_1B receptor. This explains the very significant loss of function of the gAT_1A, and the intermediate decrease in losartan affinity of the gAT_1B compared with the hAT₁ receptor.

Losartan binding was severely reduced when we replaced Val¹⁰⁸ with Ser, a polar amino acid, suggesting the need for a hydrophobic residue at position 108. Val¹⁰⁸ may be near a general nonpeptide binding site on the AT₁ receptor, providing a hydrophobic interaction that stabilizes the nonpeptidic ligands. However, the size of the hydrophobic group seems to be important; replacing Val¹⁰⁸ with an amino acid with larger hydrophobic R groups (Ile) reduced losartan binding more than a mutation with an amino acid with a smaller hydrophobic R group (Ala). Losartan may be unable to take full advantage of the hydrophobic interaction of Ile because of steric hindrance; there are four methyl groups in Ile compared with three in Val. Thus, it seems that the binding site of the nonpeptide antagonists requires nonpolar residues in the TM domains (Schambye et al., 1994). The observation that the binding characteristics of the S108V mutant of the gAT_1A receptor and those of the mutant S109Thr of the rAT_1A receptor (Ji et al., 1995) are similar supports this observation.

The role of TM III in losartan binding may not be limited to that of positions 107 and 108. Another TM III residue reported to play an important role in losartan binding is Asn¹¹¹ (Groblewski et al., 1995; Monnot et al., 1996; Groblewski et al., 1997). These residues are in the same position, based on sequence comparison analysis, as residues [such as Glu¹¹³, His¹⁰⁸, Lys¹⁸² (Schwartz, 1994), and Asp¹¹³ (Strader et al., 1989)] that play important roles in ligand interaction in other G-protein-coupled receptors, highlighting their considerable structural homology.

Leu¹⁹⁵ in TM V is also important for losartan binding in both gerbil and human AT₁ receptors. The role of Leu¹⁹⁵ is only revealed when in combination with mutations in positions 107 and 108. The effect of L195M is not only cumulative with that of S107G and/or V108I but potentiates their effect, as revealed by the introduction of the mutant V108I into the gAT_1B receptor, that of L195M into the AT_1B receptor, and by the combinatorial loss-of-function mutations in the hAT₁ receptor.

We present evidence here suggesting that the effects of positions 107, 108, and 195 might be influenced by other nonconserved amino acid residues not analyzed in the present study and that may affect the receptor conformation. For example, the gain of function of mutation G107S is different in the gAT_1A (10-fold) and gAT_1B (3-fold). The losartan affinity of the G107S/M195L double mutant gAT_1B receptor was still 3-fold lower than that of the hAT₁ receptor. Furthermore, the addition of the L195M mutant to the gAT_1A receptor decreased losartan affinity by 3.5-fold, whereas the addition of mutant V108I to the gAT_1B receptor reduced affinity for losartan 49-fold.

In addition to the interaction of selected amino acids in TM III and TM V, we show that mutations in TM II (V83L), TM IV (V150I and V151I), and the extracellular loop 2 (S177I) can affect losartan binding. These mutations have a relatively small effect on losartan binding when single but different and sometimes opposite effects when combined with other major mutations, such as G107S, I108V, and/or L195M.

There are reports on additional residues in other TM domains that may affect losartan binding. A natural mutation in TM IV, T163A, is probably responsible for the lower affinity for losartan in bovine, canine, ferret, and porcine receptors (Sasaki et al., 1991; Yoshida et al., 1992; Itazaki et al., 1993; Burns et al., 1994; Ji et al., 1995; Gosselin et al., 2000). In the turkey AT₁ receptor, the V163A mutation occurs in combination with mutations S107G and I108V, and this triple combination is probably responsible for the very low losartan affinity of the tAT₁ receptor, lower than that of gAT_1A receptor and 670-fold lower than that of rAT_1B (Murphy et al., 1993). These observations indicate that Ala¹⁶³ in TM IV is important for losartan binding, and its effect may be enhanced by mutations in TM III. However, in both gAT_1A and gAT_1B, Ala¹⁶³ is well conserved.

The K199A mutation in TM V decreased losartan binding (Ji et al., 1995; Monnot et al., 1996). In addition, there are reports in the literature of residues important for losartan binding in TM VI (Ser²⁵²) (Ji et al., 1994, 1995; Nirula et al., 1996). According to the results from experiments of human and amphibian AT₁ chimerical receptors, the TM VII (particularly Asn²⁹⁵) could also be an important region for losartan binding affinity without affecting Ang II binding (Schambye et al., 1994).

Our results and those of the literature, taken together, indicate that the area surrounded by TM III (Gly¹⁰⁷, Ile¹⁰⁸, Ser¹⁰⁹, Asn¹¹¹, and Ser¹¹⁵), TM IV (Thr¹⁶³), TM V (Met¹⁹⁵ and Lys¹⁹⁹), TM VI (Ser²⁵²), and TM VII (Asn²⁹⁵) could form a recession "pocket" discriminating between nonpeptidic ligands, such as losartan, and peptidic, such as Ang II. Our failure to significantly affect Ang II binding in the present experiments further supports this hypothesis. We interpret our results as suggesting that amino acids at positions 83, 107/108, 150/151, 177, and 195 are spatially proximate and they must be interacting with each other to influence losartan binding. Despite their locations on different transmembrane domains, most of these residues are positioned within a small distance of each other within the plasma membrane, suggesting that losartan binds to the mammalian AT₁ receptor in a plane that is one or two -helical turns below the membrane surface. Such a presumed binding pocket for the biphenylimidazole AT₁ antagonists can be formulated as a two-dimensional representation (Schambye et al., 1994) or, based on such physicochemical properties of amino acid sequences as hydrophobicity and charges, as a helical wheel diagram (Murgolo et al., 1996), predicting that most positions studied here would be nearby, facing the interior of the transmembrane bundle.

The final analysis of all amino acid residues important for binding affinity of nonpeptidic antagonists is not complete. Our studies reveal some nonconserved residues that determine the molecular requirements for biphenylimidazole recognition. However, these have been reported to be not identical to nonconserved residues necessary for high-affinity binding to AT₁ antagonists from the imidazoleacrylic class (Nirula et al., 1996).

Notwithstanding, our results are noteworthy for several reasons. First, the study of a natural mutation with substantially decreased affinity for receptor antagonists provides the advantage of maintaining a close general homology with the human AT₁ receptor with minimum three-dimensional distortion. Second, precise loss-/gain-of-function studies provided evidence that, in addition to position 108, there is a significant role for positions 107 and 195 in antagonist binding in the hAT₁ receptor.

In conclusion, we found, for both human and gerbil receptors, that the most important amino acid for losartan affinity was located in position 108 in TM III, naturally mutated in gAT_1A but conserved in gAT_1B receptors. The second most important amino acid was located in position 107 in TM III, naturally mutated in both the gAT_1A and gAT_1B. The effect of mutations in positions 107 and 108 was additive. This explains the very significant loss of function of the gAT_1A, and the intermediate affinity for losartan of the gAT_1B compared with the hAT₁ receptor. In position 108, hydrophobic residues are important, and their size may be critical for optimum losartan binding. An additional very important amino acid was located in position 195 in TM V, mutated in the gAT_1B but conserved in the gAT_1A receptor. The effect of position 195 was revealed only when the L195M mutant was combined with the G107S and/or I108V mutants, and the effects are proportionally higher when the mutants are combined. In addition, we found that, in the gerbil, some additional mutations in positions 83 in TM II, 150/151 in TM IV, and 177 in intracellular loop 2 could, when single, not affect or slightly increase losartan affinity, and, when combined with the G107S/I108V mutants, lose this property and even decrease losartan binding. Advances in the understanding of the molecular requirement for human AT₁ receptor binding to nonpeptidic antagonists could help in the development of potent and specific compounds of relevant clinical use.