©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Critical Role of a Conserved Intramembrane Tyrosine Residue in Angiotensin II Receptor Activation (*)

László Hunyady (§) , Márta Bor , Tamás Balla , Kevin J. Catt (¶)

From the (1) Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The rat type 1a (AT) angiotensin II (Ang II) receptor contains a highly conserved tyrosine residue in the fifth transmembrane region that is present in most G protein-coupled receptors. The role of this amino acid in ATreceptor activation was analyzed in a mutant receptor (Y215F) created by replacing Tyrwith phenylalanine. The mutant receptor was highly expressed in transfected COS-7 cells, and its binding affinity for the peptide antagonist [Sar,Ile]Ang II was similar to that of the wild type receptor. Although the structural integrity of the peptide ligand binding domain was preserved in the Y215F mutant receptor, its affinity for the native agonist, Ang II, was significantly reduced. Also, whereas guanosine 5`-3- O-(thio)triphosphate markedly reduced Ang II binding to the wild type receptor, it had little effect on agonist binding to the mutant receptor. Agonist-induced internalization of the mutant receptor was also impaired, and its ability to mediate inositol phosphate responses to Ang II stimulation was abolished. The concomitant decreases in receptor internalization and G protein-mediated signaling of the Y215F mutant receptor indicate that Tyrhas a critical role in ATreceptor activation. In view of its conservation among members of the seven transmembrane domain receptor superfamily, this residue is likely to be of general importance in signal transduction from G protein-coupled receptors.


INTRODUCTION

The rat type 1a (AT)() Ang II receptor is a heptahelical, G protein-coupled receptor and is widely distributed in cardiovascular, neural, and endocrine cells (1, 2) . Binding of Ang II to the ATreceptor is followed by phospholipase C activation and Casignaling via the Gfamily of G proteins (2, 3, 4) . Although all G protein-coupled receptors share the basic seven-transmembrane domain structure, relatively few amino acids are highly conserved among the numerous receptors of this type (Fig. 1). One of these conserved amino acids is a tyrosine residue located in the fifth transmembrane helix, adjacent to the amino-terminal end of the third cytoplasmic loop. The location of this amino acid, which is Tyrin the rat ATreceptor, suggests that it might play an important role in the receptor activation process. The amino-terminal region of the third cytoplasmic loop adjacent to this residue has been shown to be important in the signal generation and internalization of several G protein-coupled receptors (5, 6, 7) . Furthermore, modeling of G protein-coupled receptors based on the low resolution crystal structure of bovine rhodopsin molecule has suggested that this residue is in molecular proximity to regions that are important in receptor activation, including the conserved acidic-arginine-aromatic (DRY) triplet of the second intracellular loop (8, 9) . We have shown previously that ATreceptor internalization and signal transduction have different structural requirements (10) . However, a 6-amino acid deletion of the amino-terminal end of the third cytoplasmic loop, which included Tyr, prevented both ATreceptor internalization and signaling responses. To evaluate the role of the highly conserved Tyrresidue in the activation process of G protein-coupled receptors, we created a mutant receptor by substituting Tyrwith phenylalanine (Y215F). This mutant receptor was expressed in COS-7 cells and its binding, internalization, and signaling properties were compared with those of the wild type ATreceptor.


Figure 1: Conserved amino acids in the rat AT receptor. The helices were positioned and the most conserved residues were selected based on a comparative study and modeling of more than 200 G protein-coupled receptors (9, 27).




EXPERIMENTAL PROCEDURES

Materials

The cDNA clone (pCa18b) of the rat smooth muscle ATreceptor subcloned into the mammalian expression vector pCDM8 (Invitrogen, San Diego, CA) was kindly provided by Dr. Kenneth E. Bernstein (1) . Culture media were from Biofluids (Rockville, MD). The Medium 199 used in these experiments was modified to contain 3.6 mM K, 1.2 mM Ca, 1 g/liter bovine serum albumin, and 20 mM HEPES. Lipofectamine was from Life Technologies, Inc. I-Ang II and I-[Sar,Ile]Ang II were obtained from Hazleton Laboratories (Vienna, VA), and [H]inositol was from Amersham Corp.

Mutagenesis and Expression of the Rat Smooth Muscle ATReceptor cDNA

A HindIII- NotI fragment of the receptor cDNA was subcloned into pcDNAI/Amp (Invitrogen, San Diego, CA) as described earlier (10) . The Y215F mutant rat ATreceptor was created using the Mutagene kit (Bio-Rad), which is based on the method of Kunkel et al. (11) . The sequence of the mutant colony was identified by dideoxy sequencing using Sequenase II (Amersham-U. S. Biochemical Corp.). The wild type and mutant ATreceptor cDNA were transiently expressed in COS-7 using Lipofectamine as described previously (12) .

Inositol Phosphate Measurements

In these experiments, the culture medium was replaced 24 h after transfection with 0.5 ml of inositol-free Dulbecco's modified Eagle's medium containing 1 g/liter bovine serum albumin, 20 µCi/ml [H]inositol, 2.5% fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin as described earlier (10) . 24 h later the cells were washed twice, incubated in inositol-free modified Medium 199 in the presence of 10 mM LiCl for 30 min at 37 °C, and stimulated with 30 nM Ang II for 20 min. Inositol phosphates were extracted and analyzed by high pressure liquid chromatography as described previously (13) .

Ang II Binding to COS-7 Cells and Membranes

48 h after transfection, I-[Sar,Ile]Ang II binding was performed with intact COS-7 cells at 4 °C as described previously (10) , and the binding parameters were determined for a one site model using the computer program LIGAND (14) . To measure binding to COS-7 cell membranes, the transfected cells were washed with ice-cold phosphate-buffered saline and scraped into 3 ml of ice-cold 10 mM Tris/HCl (pH 7.4), 1 mM EDTA, then lysed by freezing on dry ice. Crude membranes were prepared by centrifuging the samples at 16,000 g. The pellet was washed in the same medium, and the protein content was determined. Binding assays were performed in 0.2 ml of binding buffer (containing 100 mM NaCl, 5 mM MgCl, 2 g/liter bovine serum albumin, and 20 mM Tris/HCl (pH 7.4)) at 25 °C. Each sample contained approximately 0.05 µCi I-Ang II or I-[Sar,Ile]Ang II, 15-25 µg of crude membranes, selected concentrations of unlabeled Ang II or [Sar,Ile]Ang II, and the indicated concentrations of GTPS. After 90-min incubation at 25 °C the unbound tracer was removed by rapid filtration and the bound radioactivity was measured by -spectrometry. The ICvalues of the displacement curves were estimated by nonlinear least squares curve fitting using the computer program ALLFIT (15) .

Receptor Internalization in Transiently Transfected COS-7 Cells

The experimental conditions for these studies were as described previously (10) . Briefly, the internalization kinetics of the mutant and wild type rat ATreceptors were measured 48 h after transfection using I-Ang II (0.05-0.1 µCi, 0.2-0.5 nM). The percent internalization at each point was calculated from the ratio of the acid-resistant (internalized) binding to the total (acid-resistant + acid-released) binding. The internalization rate was calculated by determining the slope of the initial linear phase of the internalization kinetic plot. Internalization rates of the wild type ATreceptor were measured over the first three minutes, and those of the Y215F mutant receptor were measured over the first 5 min.


RESULTS

Expression and Signal Transduction of ATReceptors

COS-7 cells were transfected with cDNA encoding either the wild type or the Y215F mutant rat ATreceptor to analyze the signaling and internalization properties of the two receptors. To determine the surface expression level and structural integrity of the Y215F receptor, binding of the Ang II antagonist analog, [Sar,Ile]Ang II, to the mutant and wild type receptors transiently expressed in intact COS-7 cells was measured at 4 °C. Scatchard analysis of the binding data showed that the expression of Y215F receptors was 69.1 ± 7.1% of that of the wild type receptor ( n = 3, Fig. 2, upper left panel). The Kof [Sar,Ile]Ang II under these conditions was 1.6 ± 0.1 nM for the wild type receptor and 1.7 ± 0.1 nM for Y215F mutant receptor ( n = 3). Despite the unimpaired affinity for [Sar,Ile]Ang II and its relatively high expression level, cells transfected with the Y215F mutant receptor showed no detectable inositol phosphate responses after stimulation with a maximally effective concentration of Ang II (Fig. 2).


Figure 2: Expression and inositol phosphate responses of Y215F mutant and wild type AT receptors in COS-7 cells. The number of binding sites for the expressed receptors was determined by Scatchard analysis of [Sar,Ile]Ang II binding data as described under ``Experimental Procedures'' ( n = 3, upper left panel). Levels of inositol phosphate ( InsP, upper right panel), inositol bisphosphate ( InsP, lower left panel), and inositol trisphosphate ( InsP, lower right panel) labeling after 30-min LiCl (10 mM) pretreatment were measured in control ( C) and Ang II ( AII, 20 min, 1 µM)-treated COS-7 cells expressing wild type and Y215F mutant rat ATreceptors. Data are shown as means ± S.E. for three experiments each performed in duplicate.



Ang II Binding to ATReceptors in COS-7 Cell Membranes

To estimate the efficiency of G protein coupling of the Y215F receptor, the binding of the native agonist, I-Ang II, was studied in membrane preparations from COS-7 cells expressing mutant and wild type ATreceptors. In contrast to its unaltered affinity for [Sar,Ile]Ang II, Y215F showed significantly reduced binding affinity for Ang II. The ICvalues for inhibition of radioligand binding by Ang II in membranes of COS-7 cells transfected with wild type and Y215F mutant ATreceptor were 0.35 ± 0.02 nM and 2.7 ± 0.1 nM, respectively ( n = 3). In addition, GTPS markedly reduced Ang II binding to the wild type receptor but had only a minor effect on agonist binding to Y215F (Fig. 3). The impairment of Ang II binding to the wild type receptor in the presence of GTPS reflected a decrease in the affinity of the receptor (data not shown), secondary to its uncoupling from G protein(s). These data, in good agreement with the inositol phosphate results, suggest that the Y215F mutant receptor has impaired ability to couple to G proteins.


Figure 3: Inhibitory effects of GTPS on Ang II binding to Y215F mutant and wild type receptors AT. Crude membranes were prepared from COS-7 cells expressing wild type () or Y215F mutant () ATreceptors. I-Ang II binding was measured in the presence of the indicated concentrations of GTPS as detailed under ``Experimental Procedures.'' The Blevels (100%) for the wild type and Y215F mutant were 7152 ± 1616 cpm and 1016 ± 186 cpm, respectively. Data are shown as means ± S.E. for three experiments each performed in duplicate.



Internalization of the Y215F Mutant ATReceptor

As reported earlier, the rat ATreceptors expressed in COS-7 cells undergo rapid internalization during agonist activation. The role of Tyrin the internalization process was studied by comparing the kinetics of I-Ang II sequestration in Y215F mutant and wild type rat ATreceptors. As shown in Fig. 4, the internalization of I-Ang II was significantly slower in cells expressing the mutant receptor than in those expressing the wild type ATreceptor (Fig. 4, left panel). This difference was particularly evident when the internalization rates were compared by estimating the slope of the initial linear phase of the time course of receptor internalization (Fig. 4, right panel).


Figure 4: Internalization of wild type () and Y215F mutant () AT receptors. The internalization kinetics ( left panel) and the initial internalization rates ( right panel) were calculated as described under ``Experimental Procedures.'' Data are shown as means ± S.E. for four experiments each performed in duplicate.




DISCUSSION

The Tyrresidue of the type 1 Ang II receptor is a highly conserved amino acid in the fifth transmembrane domain of numerous G protein-coupled receptors (8, 9) . Recent modeling data have suggested that this tyrosine residue clusters with the highly conserved DRY sequence located at the end of helix 3 and that this region is a likely site for interaction with G proteins (8) . While the importance of the DRY sequence at the bottom of helix 3 has been established for the ATreceptor (16) as well as for other G protein-coupled receptors (5, 6) , the present study provides experimental evidence for a role of the conserved Tyrresidue in receptor activation and signal transduction. Although further studies are required to clarify its exact function, our data, together with the above-mentioned modeling studies, indicate the importance of this conserved residue in the activation process of G protein-coupled receptors. Earlier studies have identified other residues in the transmembrane domains as well as in the second and third intracellular loops that are critical for receptor activation (5, 6, 7, 17, 18, 19, 20, 21) . Although the intramembrane residues are probably important for the active conformation of the receptor, the residues located in the intracellular domains are more likely to participate in G protein coupling. In the m3 muscarinic receptor, which also couples to the Gfamily of G proteins, a tyrosine residue located 4 residues downstream of the conserved tyrosine residue in the fifth transmembrane domain (corresponding to Tyr) was found to be important for inositol phosphate signaling (19) . Since this distance is approximately one turn in an -helical structure, it is possible that Tyrparticipates in propagation of the agonist-induced conformational change from the binding site to the intracellular loops.

The type 1 Ang II receptors, like other G protein-coupled receptors, undergo rapid endocytosis following agonist binding (22, 23, 24) . Although receptor internalization and signaling both require the active conformation of the receptor, the structural determinants of the internalization and signaling processes are not identical (6, 10, 25) . For example, we have recently identified a sequence in the cytoplasmic tail of the rat ATreceptor that is a critical determinant of receptor internalization, but has no significant role in Ang II-induced signal transduction (12) . Conversely, receptors with impaired signaling ability have been shown to undergo rapid endocytosis (10, 26) . In this context it is important to note that the internalization rate of the Y215F receptor was substantially reduced, but unlike the inositol phosphate response, was readily demonstrable. Despite the parallel impairment of the internalization and signaling properties of the Y215F mutant receptor, replacement of this single amino acid did not cause a major perturbation of receptor structure as indicated by its retention of high affinity for the peptide antagonist, [Sar,Ile]Ang II. The concomitant loss of agonist binding affinity was consistent with the impaired ability of the mutant receptor to interact with G proteins.

The data presented in this paper provide direct evidence for the importance of the highly conserved tyrosine residue located in the cytoplasmic aspect of the fifth transmembrane domain in the activation process of a heptahelical receptor. These data are in accordance with the predicted role of this residue based on its location in modeling studies of G protein-coupled receptors. The parallel impairment of the internalization, signaling, and G protein coupling of the mutant ATreceptor suggest that Tyris a crucial determinant of multiple aspects of its activation mechanism.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Permanent address: Dept. of Physiology, Semmelweis University School of Medicine, P. O. Box 259, H-1444 Budapest, Hungary.

To whom correspondence should be addressed: ERRB, NICHD, National Institutes of Health, Bldg 49, Rm. 6A36, Bethesda, MD 20892-4510. Tel.: 301-496-2136; Fax: 301-480-8010.

The abbreviations used are: ATreceptor, type 1 angiotensin II receptor; Ang II, angiotensin II; GTPS, guanosine 5`-3- O-(thio)triphosphate.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.