©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Conserved NPLFY Sequence Contributes to Agonist Binding and Signal Transduction but Is Not an Internalization Signal for the Type 1 Angiotensin II Receptor (*)

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

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A conserved NPXY sequence that is located in the seventh transmembrane helix of many G protein-coupled receptors has been predicted to participate in receptor signaling and endocytosis. The role of this sequence (NPLFY) in angiotensin II receptor function was studied in mutant and wild-type rat type 1a angiotensin II receptors transiently expressed in COS-7 cells. The ability of the receptor to interact with G proteins and to stimulate inositol phosphate responses was markedly impaired by alanine replacement of Asn and was reduced by replacement of Pro or Tyr. The F301A mutant receptor exhibited normal G protein coupling and inositol phosphate responses, and the binding of the peptide antagonist, [Sar,Ile]angiotensin II, was only slightly affected. However, its affinity for angiotensin II and the nonpeptide antagonist losartan was reduced by an order of a magnitude, suggesting that angiotensin II and losartan share an intramembrane binding site, possibly through their aromatic moieties. None of the agonist-occupied mutant receptors, including Y302A and triple alanine replacements of Phe, Tyr, and Phe, showed substantial changes in their internalization kinetics. These findings demonstrate that the NPLFY sequence of the type 1a angiotensin II receptor is not an important determinant of agonist-induced internalization. However, the Phe residue contributes significantly to agonist binding, and Asn is required for normal receptor activation and signal transduction.


INTRODUCTION

The type 1 (AT)()receptor for the vasoactive peptide Ang II is a member of the family of seven transmembrane domain receptors(1, 2) . The AT receptor has been reported to interact with several G proteins, including G, G, and G, but its major physiological functions are expressed through G-mediated activation of phospholipase C followed by phosphoinositide hydrolysis and Ca signaling(1, 2, 3, 4, 5) . During the last decade, hundreds of G protein-coupled receptors and their subtypes have been cloned and sequenced(6, 7) . Although these receptors all possess the basic seven-transmembrane structure, the number of highly conserved amino acids shared by the superfamily of G protein-coupled receptors is relatively few(6, 8, 9) . One such conserved motif is the characteristic NPXY sequence that is located in the seventh transmembrane domain of most receptors, and in the rat smooth muscle AT receptor is Asn-Pro-Leu-Phe-Tyr (10) (Fig. 1).


Figure 1: Amino acid sequence of the Ang II molecule and localization of the conserved NPXY sequence in the seventh transmembrane domain of the rat AT receptor. The position of the amino acids is shown based on a recently published model of the human -adrenergic receptor (9).



Several G protein-coupled receptor models have been based on the structure of bacteriorhodopsin. This heptahelical membrane protein shares no sequence homology with mammalian G protein-coupled receptors, but it has an identical folding pattern and functional resemblance to mammalian opsins(11) . The recently reported low resolution structural image of bovine rhodopsin (12) has made it possible to further improve these models(9, 13) . A recent model of aminergic G protein-coupled receptors suggests that the NPXY sequence is ideally placed to receive a signal from agonist-induced conformational changes in the ligand binding region(9, 14) . This sequence is in close proximity to the functionally important asparagine-aspartic acid pair located in transmembrane segments 1 and 2 and may participate in important hydrogen bonding interactions. Most of the available models also predict that the highly conserved intramembrane proline residues, which disrupt the -helical structure of the transmembrane domains, serve as hinges that participate in the agonist-induced conformation change of G protein-coupled receptors(9, 11) . One such proline residue is located within the seventh transmembrane domain in the conserved NPXY sequence, and this residue (Pro) of the m muscarinic receptor has been found to be important for signal transduction(15) .

Another interesting feature of the NPXY sequence is its similarity to the NPXY internalization sequence that was first described in the cytoplasmic segment of the low density lipoprotein receptor(16) . In recent studies, the tyrosine residue in this sequence was found to be essential for sequestration of the -adrenergic receptor (17) but not for the internalization of the gastrin-releasing peptide receptor(18) . The gastrin-releasing peptide receptor and the AT receptors, unlike the -adrenergic receptor, contain an additional aromatic amino acid (Phe and Phe, respectively) in their NPXY sequences(1, 18) . The presence of such residue might be important since phenylalanine can substitute for the tyrosine residue in the NPXY internalization sequence of nutrient receptors(16) . The present study was performed to analyze the role of the NPXY sequence in ligand binding, internalization, and signaling of G protein-coupled receptors, utilizing the rat AT receptor as a model to evaluate its participation in these critical aspects of receptor function.


EXPERIMENTAL PROCEDURES

Materials

The cDNA clone (pCa18b) of the rat smooth muscle AT receptor subcloned into the mammalian expression vector pCDM8 (Invitrogen, San Diego, CA) was kindly provided by Dr. Kenneth E. Bernstein(10) . Restriction enzymes were obtained from Boehringer Mannheim or New England Biolabs (Beverly, MA). 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. Lipofectin, lipofectamine and Opti-MEM I were from Life Technologies, Inc. Losartan was a gift from Dr. P. C. Wong (DuPont, Wilmington, DE). I-Ang II and [I-Sar,Ile]Ang II were obtained from Hazleton Laboratories (Vienna, VA) or DuPont NEN; [H]inositol was from Amersham Corp.

Mutagenesis of the Rat Smooth Muscle ATReceptor cDNA

The rat AT receptor cDNA was subcloned into the mammalian expression vector pcDNAI/Amp (Invitrogen) as described earlier(19) . Mutant rat AT receptors were created using the Mutagene kit (Bio-Rad, Hercules, CA), which is based on the method of Kunkel et al.(20) . Each mutant contained a silent restriction site to facilitate the screening of colonies. Oligonucleotides were obtained from Midland Certified Reagent Co. (Midland, TX). All mutations were verified by dideoxy sequencing using Sequenase II (U. S. Biochemical Corp.).

Transient Transfection of COS-7 Cells

COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 2 mML-glutamine, 10% heat-inactivated fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin. To determine inositol phosphate responses, internalization kinetics, or [Sar,Ile]Ang II binding to intact cells, the cells were seeded in 24-well plates (50,000 cells/well) 3 days before transfection. Transient transfection was performed by replacing the culture medium with 0.5-ml aliquots of Opti-MEM I containing 8 µg of lipofectamine and 1 µg of plasmid DNA/well, or with increasing amounts of cDNA as indicated in the legend to Fig. 4. The cells were incubated for 5-6 h in this solution, and the medium was replaced with culture medium. For membrane binding studies, 10 cells were grown on 100-mm culture dishes for 3 days. Transfections were performed for 5-6 h using 5 ml of Opti-MEM I containing 16 µg/ml lipofectamine and 10 µg of plasmid DNA. After transfection, the medium was replaced with the culture medium. All experiments were performed 48 h after the initiation of the transfection procedure.


Figure 4: Correlation between AT receptor expression level and the amplitude of inositol phosphate responses. COS-7 cells were plated in 24-well plates and transfected with increasing quantities of wild-type AT receptor DNA (0.03-2.5 µg) using lipofectamine (16 µg/ml) or lipofectin (10 µg/ml). For inositol phosphate measurements, the cells were prelabeled for 24 h with [H]inositol and stimulated with 1 µM Ang II in the presence of 10 mM LiCl. The extracellular Ang II receptor expression level was measured by analyzing [I-Sar,Ile]Ang II displacement curves. Methodological details are described under ``Experimental Procedures.'' The combined InsP and InsP responses are shown as means ± range of duplicates from a representative experiment of three similar observations.



[Sar,Ile]Ang II Binding to Intact Cells

To determine the expression level and structural integrity of the mutant receptor, the number of Ang II binding sites was determined by incubating the transfected cells with [I-Sar,Ile]Ang II (0.05-0.1 µCi/sample) and increasing concentrations of unlabeled [Sar,Ile]Ang II in Medium 199 (HEPES) for 6 h at 4 °C. The cells were washed twice with ice-cold Dulbecco's phosphate-buffered saline, and the radioactivity associated with the cells was measured by -spectrometry after solubilization with 0.5 M NaOH, 0.05% SDS. The displacement curves were analyzed with the Ligand computer program using a one-site model(21) .

Binding to COS-7 Cell Membranes

48 h after transfection, the cells were washed and scraped into 1.5 ml of ice-cold 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, and lysed by freezing. Crude membranes were prepared by centrifuging the samples at 16,000 g. The pellet was resuspended in binding buffer (containing 100 mM NaCl, 5 mM MgCl, and 20 mM Tris-HCl (pH 7.4)), and the protein content was determined. Binding assays were performed in 0.2 ml of binding buffer supplemented with 2 g/liter bovine serum albumin at 25 °C. Each sample contained 0.05-0.1 µCi of I-Ang II or [I-Sar,Ile]Ang II, 15-30 µg of crude membranes, and the indicated concentrations of unlabeled [Sar,Ile]Ang II, losartan, or Ang II in the presence or absence of 5 µM GTPS as indicated. After a 90-min incubation at 25 °C, the unbound tracer was removed by rapid filtration, and the bound radioactivity was measured by -spectrometry.

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(19) . After 24 h of labeling, the cells were washed twice and incubated in inositol-free modified Medium 199 in the presence of 10 mM LiCl for 30 min at 37 °C, and then stimulated with 30 nM Ang II for 20 min or, in the case of F301A, with 1 µM Ang II for 20 min. Incubations were stopped by adding perchloric acid (5% (v/v) final concentration). Inositol phosphates were extracted and analyzed by high performance liquid chromatography as described previously(22) .

Internalization of Wild-type and Mutant ATReceptors

Before each experiment the medium was replaced by Hepes-buffered Medium 199. To determine the internalization kinetics of the mutant and wild-type AT receptors I-Ang II (0.05-0.1 µCi) was added in the same medium, and the cells were incubated at 37 °C for the indicated times. Incubations were stopped by placing the cells on ice and rapidly washing them twice with 1 ml of ice-cold Dulbecco's phosphate-buffered saline. The cells were incubated for 10 min in 0.5 ml of acid wash solution (150 mM NaCl, 50 mM acetic acid) to remove the surface-bound radioligand. The supernatant containing the acid-released radioactivity was collected, and the cells were treated with 0.5 M NaOH and 0.05% SDS to solubilize the acid-resistant (internalized) radioactivity. The radioactivities were measured by spectrometry, and the percent of internalized ligand at each time point was calculated from the ratio of the acid-resistant binding to the total (acid-resistant + acid-released) binding.

To determine the internalization kinetics of prelabeled receptors, the cells were incubated with I-Ang II (0.05-0.1 µCi) in 0.25 ml Medium 199 for 3-4 h at 4 °C to permit binding in the absence of receptor internalization. The unbound tracer was removed by washing the cells twice with 1-ml aliquots of ice-cold Dulbecco's phosphate-buffered saline. After addition of 0.5 ml of warm (37 °C) Medium 199, the cells were incubated for the indicated times at 37 °C to allow internalization to proceed. Incubations were stopped by placing the cells on ice, and the medium containing the tracer released during the incubation was collected and replaced with 0.5 ml of ice-cold acid wash solution. The extracellular (acid-sensitive) and internalized tracer were measured as described above. The total binding was calculated as the sum of the released (into the medium), extracellular (acid-sensitive), and internalized (acid-resistant) radioactivities. The internalized or released radioactivity was expressed as a percent of the total binding at each time point.


RESULTS

Expression of Mutant ATReceptors in COS-7 Cells

[Sar,Ile]Ang II binding was measured in intact COS-7 cells transfected with mutant or wild-type AT receptors to determine the expression level and the functional integrity of these receptors at the plasma membrane. As shown in , all mutant receptors analyzed in this study bound the antagonist radioligand with high affinity. It is interesting to note that all mutants in which Phe was replaced (F301A, L300A/F301A, F301A/Y302A, F301A/Y302A/F304A) showed slightly but consistently lower binding affinity than the other mutants or the wild-type receptor. The expression levels of the mutant receptors showed more significant variations. While alanine replacement for Asn and Tyr had no major effect on receptor expression, mutation of the three inner residues (Pro, Leu, and Phe) reduced expression to approximately one-third of that of the wild-type receptor (). The effect of amino acid replacement on receptor expression appeared to be additive, since L300A/F301A showed further reduction compared with L300A and F301A, and F301A/Y302A and F301A/Y302A/F304A showed progressively reduced expression levels compared with F301A or Y302A.

Ang II Binding to COS-7 Cell Membranes Expressing Wild-type or Mutant ATReceptors

Previous reports showed that the type 1 Ang II receptors can couple to multiple G proteins including G, G, and G (1). To evaluate the ability of the AT receptor to couple to G proteins, we measured the effect of GTPS on Ang II binding in COS-7 cell membranes. Addition of GTPS (5 µM) caused a 3.45 ± 0.38-fold (n = 3) decrease in the affinity of the wild-type receptor for Ang II, indicating that the expressed AT receptor is coupled to G proteins. In the presence of the GTP analogue, when the receptor is uncoupled from G proteins, the binding affinities of the N298A, P299A, L300A, and Y302A mutants for Ang II were similar to that of the wild-type receptor (). In the absence of GTPS, the affinities of the mutants for Ang II were lower than that of the wild-type receptor, suggesting that receptor-G protein interaction is affected in these mutants. The GTPS-induced change in affinity was most impaired in the N298A and P299A mutant receptors, but it was also evident in the Y302A mutant (Fig. 2). Interestingly, despite its near normal affinity for [Sar,Ile]Ang II (), the F301A mutant receptor had significantly reduced affinity for the physiological agonist, Ang II ().


Figure 2: Effect of GTPS (5 µM) on the binding affinity of Ang II for the wild-type (w.t.), N298A (N), P299A (P), L300A (L), F301A (F), and Y302A (Y) mutant receptors. The GTPS-induced change in the receptor affinity is shown as percent of the shift measured for the wild-type receptor. GTPS caused a 3.4-fold (100%) increase in the IC of the wild-type receptor (n = 3). For the F301A mutant [I-Sar,Ile]Ang II was used as tracer since the I-Ang II labeling of this receptor was low. Under these conditions GTPS caused a 1.9 ± 0.2-fold (100%) increase in the IC of Ang II for the wild-type receptor (n = 3). Data are shown as means ± S.E. for three experiments performed in duplicate.



Displacement of [I-Sar,Ile]Ang II in COS-7 Cell Membranes Expressing F301A or Wild-type ATReceptors

Since both the expression levels () and the agonist affinity () of the F301A receptor were impaired, it was difficult to accurately characterize this mutant using I-Ang II as radioligand. For this reason, [I-Sar,Ile]Ang II was utilized to measure the binding properties of F301A. Consistent with the data obtained in intact cells, the F301A mutant receptor showed only slightly decreased affinity relative to the wild-type receptor when [Sar,Ile]Ang II was used to displace the radioligand (IC: wild-type, 0.53 ± 0.09 nMversus F301A, 0.92 ± 0.05 nM; n = 3) (Fig. 3, upper panel). In contrast, the ability of the agonist ligand (Ang II) to displace [I-Sar,Ile]Ang II was reduced 9.7 ± 0.9-fold in the F301A mutant (IC: wild-type, 3.0 ± 0.1 nMversus F301A, 29.5 ± 2.6 nM; n = 3) (Fig. 3, middle panel). This selective reduction of Ang II binding affinity was not related to impaired G protein coupling, since the effect of GTPS on binding was similar to that observed in the wild-type receptor (Fig. 2). The nonpeptide AT receptor antagonist losartan inhibited [I-Sar,Ile]Ang II binding to the wild-type receptor with an IC of 26.6 ± 1.4 nM (n = 3). However, the affinity of the F301A mutant receptor for losartan was reduced 9.9 ± 2.5-fold (IC, 263 ± 66 nM; n = 3) (Fig. 3, lower panel), similar to its loss of affinity for the native agonist, Ang II.


Figure 3: [I-Sar,Ile]Ang II binding of the F301A mutant () and wild-type () rat AT receptors. The tracer was displaced with the indicated concentrations of [Sar,Ile]Ang II (upper panel), Ang II (middle panel), or losartan (lower panel). Data are shown as means ± S.E. for three experiments performed in duplicate.



Effects on Inositol Phosphate Signaling

To determine the ability of the mutant receptors to couple to phospholipase C via G and related proteins, we measured the inositol phosphate response of transfected COS-7 cells to Ang II in the presence of LiCl. As reported earlier, under these experimental conditions the major accumulating products of phosphoinositide hydrolysis are InsP and InsP in AT receptor-transfected COS-7 cells(19) . Since the expression levels of the mutant AT receptors showed significant variations (), the relationship between receptor expression and the amplitude of the maximal inositol phosphate response was determined after transfecting COS-7 cells with increasing amounts of the wild-type AT receptor cDNA. Despite the wide range of receptor expression in such cells, there was a linear relationship (r = 0.98) between the measured extracellular receptor sites and the inositol phosphate responses to agonist stimulation (Fig. 4). This finding indicates that valid comparisons between cells expressing mutant AT receptors can be made by normalizing their inositol phosphate responses to the number of plasma membrane binding sites (Fig. 5, lower panel).


Figure 5: Ang II-induced inositol phosphate responses of wild-type (w.t.), N298A (N), P299A (P), L300A (L), F301A (F), and Y302A (Y) mutant AT receptors. Cells were prelabeled for 24 h with [H]inositol and preincubated in the presence of 10 mM LiCl as described under ``Experimental Procedures.'' The H radioactivity of InsP (upper panel) or InsP (middle panel) fractions after a 20-min treatment with (hatchedbars) or without (openbars) Ang II in the presence of 10 mM LiCl is shown. Maximally active concentration of 30 nM Ang II was used for each receptor except for F301A where 1 µM Ang II was used. The lower panel shows the combined InsP + InsP responses normalized to the number of I-[Sar,Ile]Ang II binding sites. These data are expressed as percent of the wild-type (w.t.) response, which was 38,600 ± 4,600 cpm/pmol binding sites (n = 3) and are shown as means ± S.E. from three independent experiments each performed in duplicate.



The inositol phosphate responses of cells expressing mutant AT receptors were measured after maximal agonist stimulation with 30 nM Ang II. However, 1 µM Ang II was added in studies on the F301A receptor due to its reduced binding affinity for the native agonist. Single alanine replacements in the NPLFY sequence resulted in mutants showing various degrees of reduction of the inositol phosphate responses. The most prominent decrease was detected in the N298A, P299A, and F301A mutants during Ang II stimulation (Fig. 5). After normalization of the data to the receptor expression level, the impaired response of cells transfected with the F301A receptor was attributable to its lower expression level, while the L300A receptor appeared to activate phospholipase C more effectively than the wild-type receptor (Fig. 5, lower panel). The most significant impairment of inositol phosphate signaling was observed in COS-7 cells expressing the N298A receptor, which showed more than 60% reduction of inositol phosphate accumulation. However, the P299A and Y302A receptors also mediated consistently reduced inositol phosphate responses (Fig. 5, lower panel).

Since binding studies revealed significant differences in the agonist affinities of the mutant AT receptors, a detailed analysis of the dose-dependence of their signaling responses was performed. The EC values for inositol phosphate responses of the wild-type and mutant AT receptors showed a good correlation with the respective IC values for inhibition of radioligand binding by native Ang II (). For example the dose-response curve for Ang II-induced inositol phosphate production of the F301A mutant receptor was shifted to the right compared with that of the wild-type receptor, consistent with the reduced agonist affinity of this mutant (Fig. 6). However, the maximum level of stimulation mediated by the F301A receptor showed no reduction when the data were normalized for the reduced number of binding sites (Fig. 5, lower panel). On the other hand, the higher EC values observed in cells expressing the N298A receptor (Fig. 6) and the P299A and Y302A receptors () were paralleled by impaired G protein-coupling. This is indicated by the reduced maximal response (Fig. 5, lower panel) and decreased GTPS effect on Ang II binding (Fig. 2) to these mutant receptors.


Figure 6: Dose-response curve of InsP (upper panel) and InsP (lower curve) responses of the wild-type (), F301A (), and N298A () rat AT receptors. Data are expressed as percent of the maximum response obtained after stimulation with 1 µM Ang II, and are representative of three similar observations. In the experiment shown, the basal and maximally stimulated levels of inositol phosphates were as follows. InsP, 598 cpm versus 9,297 cpm for the wild-type AT receptor, 651 versus 2,433 cpm for the N298A receptor, and 451 cpm versus 2,879 cpm for F301A receptor; InsP, 264 cpm versus 2,321 cpm for the wild-type receptor, 300 versus 1,092 cpm for the N298A, and 268 cpm versus 929 cpm for the F301A receptor.



Internalization of Wild-type and Mutant ATReceptors in COS-7 Cells

The AT receptor expressed in COS-7 cells undergoes rapid agonist-induced internalization, similar to that of the native receptors of smooth muscle, adrenal glomerulosa, and other cell types(2, 19, 23, 24) . Single alanine replacements in the NPLFY sequence caused relatively minor impairment of the internalization kinetics of the hormone-receptor complex (Fig. 7). While the rates of internalization of the N298A, F301A, and Y302A receptors were somewhat slower than that of the wild-type receptor, all mutant receptors showed rapid agonist-induced internalization, and the quantity of internalized radioligand exceeded that of extracellular binding after a 30-min incubation at 37 °C (Fig. 7).


Figure 7: Internalization kinetics of wild-type (), N298A (), P299A (, upper panel), L300A (), F301A (, middle panel), and Y302A (, lower panel) AT receptors in the continuous presence of I-Ang II. The dashedline represents the wild-type curve on the lower panels. Results are expressed as percent of the total binding for each time point. Data are shown as means ± S.E. for three experiments performed in duplicate.



In the low density lipoprotein receptor, the tyrosine residue of the NPXY motif can be replaced by phenylalanine with no loss of function of the internalization signal(16) . Since the AT receptor Tyr is preceded by a phenylalanine, the Y302A mutant contains an NPLF sequence that meets the criteria of an internalization signal. To evaluate the possibility that the neighboring phenylalanine residues could substitute for Tyr when the latter is replaced with alanine, double (F301A/Y302A) and triple (F301A/Y302A/F304A) alanine replacement mutants were analyzed. Like the F301A mutant receptor, these mutants showed reduced expression levels and slightly reduced antagonist binding () but exhibited markedly impaired agonist binding (data not shown). However, these mutant receptors underwent rapid agonist-induced endocytosis, indicating that the NPXY sequence is not a major determinant of the internalization of the AT receptor (Fig. 8).


Figure 8: Internalization kinetics of wild-type (), F301A/Y302A () and F301A/Y302A/F304A () mutant receptors in the continuous presence of I-Ang II. Results are expressed as percent of the total binding for each time point. Data are shown as means ± S.E. for three experiments performed in duplicate.



Internalization Kinetics in Prelabeled Cells

To further analyze the role of Tyr in the endocytosis of AT receptors, internalization kinetics were measured in cells prelabeled with I-Ang II. Prelabeling was performed at 4 °C to prevent internalization of the radioligand as described under ``Experimental Procedures.'' After warming the cells to 37 °C, more than 60% of the tracer bound to the wild-type receptor internalized within 5 min (Fig. 9, upper panel) similar to the previously reported rapid kinetics of endogenous AT receptors(23, 24) . At the same time (5 min), less than 20% of the radioactivity was released into the medium (Fig. 9, lower panel), indicating that dissociation of the agonist from the receptor is relatively slow in comparison with the rapid kinetics of the internalization process. The release of bound and internalized radioactivity into the incubation medium exhibited biphasic kinetics. As shown for the wild-type receptor (Fig. 9, lower panel), the initial phase of release, which is largely due to dissociation of the surface-bound ligand, began to plateau by 5 min, reflecting the concomitant decrease in surface-binding by 5 min. After 5 min, the release showed a second increase that was associated with a progressive decrease in intracellular labeling. This radioactivity must be derived from recycling of the internalized tracer, since by 5 min, the extracellular binding was reduced to 20% of its initial value, and the internalized radioactivity is the only pool that can account for the second phase of release into the medium (Fig. 9).


Figure 9: Internalization and release kinetics of I-Ang II in prelabeled COS-7 cells. COS-7 cells were transfected with wild-type (), Y302A mutant (), or combined F301A/Y302A/F304A mutant () rat smooth muscle AT receptor. Cells were prelabeled with I-Ang II at 4 °C, and kinetics of the Ang II internalization (upper panel) and release of the tracer into the media (lower panel) were measured at 37 °C as described under ``Experimental Procedures.'' Data are expressed as percent of the total binding, and shown as means ± S.E. for three independent experiments.



The initial rate of internalization of the Y302A mutant receptor was slightly reduced under these conditions (Fig. 9, upper panel) in accordance with the slower initial kinetics detected in the continuous presence of the radiolabeled agonist (Fig. 7). The Y302A mutant also displayed a moderate decrease in the dissociation and recycling kinetics (Fig. 9), indicating that the turnover of this receptor is modestly impaired. The F301/Y302A/F304A mutant AT receptor showed more rapid dissociation, which is consistent with the lower affinity of this receptor (Fig. 9, lower panel). The initial rate of internalization of this combined mutant receptor showed a similar minor impairment as the Y302A mutant (Fig. 9, upper panel). Despite the modest impairment of the receptor internalization kinetics of the Tyr mutant receptors, the above data are not consistent with the hypothesis that the NPLFY sequence of the AT receptor serves as an internalization signal in the same manner as the NPXY sequence of the low density lipoprotein receptor.


DISCUSSION

Detailed analysis of the NPLFY sequence in the seventh transmembrane domain of the rat AT receptor has revealed that, in addition to its structural role, this region is also an important functional determinant of receptor expression, affinity, and signal transduction. Alanine replacement of each amino acid, particularly Pro, Leu, and Phe, caused a reduction in the number of extracellular binding sites. While the affinity of the expressed mutant receptors for the peptide antagonist [I-Sar,Ile]Ang II showed only minor variations, the affinity of the F301A mutant AT receptor for the native agonist, Ang II, and the nonpeptide antagonist, losartan, was markedly reduced. This finding is of interest since Ang II contains aromatic residues that are critical for its binding to the receptor.

As discussed above, recent modeling studies of G protein-coupled receptors have suggested that the NPXY sequence has an important role in the agonist-induced conformation change that leads to receptor signaling(9, 14) . In agreement with this hypothesis, alanine replacement of Asn in the rat AT receptor markedly reduced both coupling to inositol phosphate production and interaction with G proteins as measured by the effect of GTPS on Ang II binding. The model also predicts that Asn is in close proximity to the conserved Asp residue in the second transmembrane helix, and may participate in important hydrogen bonding interactions. The demonstrated importance of Asp in AT receptor signaling (19, 25) is consistent with the role of the corresponding residue in the activation of many other G protein-coupled receptors(8) . Although a recent study suggested that Asp interacts with Tyr in the AT receptor (26), more recent models relying on the recently published low resolution crystal structure of bovine rhodopsin have suggested that Asn is in an appropriate location to participate in such an interaction(9, 13, 14) . The present data are in good agreement with this prediction. It is interesting to note that the gonadotropin-releasing hormone receptor contains an Asn in the second transmembrane domain and an Asp in the seventh intramembrane helix, an arrangement that may represent a natural reciprocal mutation of these conserved residues(27) .

The functional importance of these residues has been demonstrated, and the existence of such a receptor supports the suggested structural and functional relationship of these residues(27) . More detailed studies will be necessary to clarify the proposed hinge function of Pro in the receptor activation process. In the present work, replacement of Pro with alanine, which would stabilize the -helical structure of the seventh transmembrane domain, caused relatively minor impairment of the inositol phosphate responses. However, the G protein coupling of this mutant receptor was significantly impaired, suggesting that Pro might be important for interaction with G proteins other than G. Similar discrepancies between the inositol phosphate response and G protein coupling of the AT receptor have been previously observed (19) and could likewise reflect the fact that the type 1 Ang II receptor interacts with multiple G proteins.

It has been reported that the tyrosine residue of the NPXY sequence is required for agonist-induced sequestration of the -adrenergic receptor(17) . Our data demonstrate that replacement of this tyrosine residue (Tyr) with alanine has only a minor effect on the internalization kinetics of the AT receptor, and similar findings were reported for the gastrin-releasing peptide receptor(18) , another Ca-mobilizing hormone receptor. However, we also examined the potential role of nearby aromatic amino acids. This was important since replacement of Tyr with alanine changes the NPLFY sequence to NPLFA, which might still serve as a fully functional internalization signal since NPXF can substitute for the NPXY sequence in the low density lipoprotein receptor(16) . In fact, the mutant AT receptor containing alanine replacements for the Phe and Tyr residues (F301A/Y302A) was well internalized, and an additional triple alanine mutant receptor, in which Phe was also replaced (F301A/Y302A/F304A) exhibited internalization kinetics even closer to those of the wild-type receptor.

The improved internalization kinetics of the triple alanine mutant receptor strengthened our conclusion that Phe and Tyr in the NPLFY sequence of the AT receptor do not participate in agonist-induced internalization of the receptor. The minor differences detected in the internalization kinetics of the mutant receptors are probably due to an overall conformational effect, perhaps affecting the position of the cytoplasmic tail of the receptor, which contains sequences essential for the agonist-induced internalization process(28) . Most of the identified sequences in G protein-coupled receptors that regulate agonist-induced endocytosis have been found to be serine-threonine rich (28-33). These observations, and the inability of the NPLFY sequence to serve as an internalization signal in the AT receptor, suggest that the endocytosis of G protein-coupled receptors is regulated by an alternative mechanism than that utilized for the tyrosine-containing signal-regulated endocytosis of nutrient and growth factor receptors.

Early structure-function studies on the Ang II molecule revealed that the aromatic side groups of Tyr and His, together with the guanidine group of Arg, are essential for the binding of the octapeptide hormone, while the phenyl group of Phe carries the information needed for the biological response(34) . The most frequently used peptide antagonists of Ang II contain sarcosine in position 1 (which increases the binding affinity of the ligand) but do not contain Phe, which is essential for activation of the receptor. The present data, in accordance with previous structure-function studies on the ligand(34) , suggest that the binding of the amino-terminal (charged) portion of the Ang II molecule, and that of the more carboxyl-terminal aromatic residues, are stabilized by different interactions and may occupy binding sites located in different regions of the receptor. Thus, the amino-terminal charged end of Ang II and its peptide antagonists is likely to interact with the recently-defined extracellular ligand binding region of the AT receptor(35) , whereas the aromatic residues of Ang II probably interact with amino acids that include residues as deeply located in the membrane as Phe.

The role of clustered aromatic amino acids in stabilization of the tertiary structure of proteins is well known(36) , and modeling studies have suggested that such clusters provide an important frame for the ligand binding site of many G protein-coupled receptors(11, 14) . Since the conserved tryptophane and phenylalanine residues in helices 4 and 6 that were suggested to provide an aromatic floor for the binding sites of aminergic receptors are also present in AT receptors, it is conceivable that aromatic interactions between the intramembrane helices and Ang II are important determinants of agonist binding. The proposed intramembrane location of the binding site that interacts with the carboxyl-terminal residues of the Ang II molecule is also in agreement with site-directed mutagenesis studies that suggested that Lys in the fifth intracellular helix of the bovine AT receptor is important for ligand binding and might interact with the carboxyl-terminal carboxyl group of the ligand(37) . The selective effect of F301A on agonist binding suggests that interactions between the aromatic residues of the hormone molecule and those of the intramembrane segment of the AT receptor are important for binding of the native agonist. The binding of [Sar,Ile]Ang II to the F301A mutant receptor is less affected since Sar-substituted Ang II peptides have increased affinity for the AT receptor due to the increased basicity of their amino-terminal region(38) , while the aromatic interaction has less effect due to the absence of the phenylalanine residue in position 8.

The finding that the F301A receptor has much greater impairment of binding affinity for Ang II and the nonpeptide antagonist losartan than for the peptide antagonist is particularly interesting. The Phe residue of the AT receptor is likewise important for the binding of losartan but not the peptide antagonist (39). The participation of this residue in both Ang II and losartan binding suggests that the binding of the agonist and the nonpeptide antagonist share an overlapping site that is likely to be stabilized by aromatic interactions. This finding accounts for the competitive nature of Ang II and losartan binding to the AT receptor. Despite its reduced affinity for Ang II, the F301A receptor showed normal G protein coupling and full inositol phosphate responses to maximal agonist stimulation. This suggests that an additional site of interaction is required for agonist activation of the receptor, in accordance with the role of Phe in both agonist and nonpeptide antagonist binding.

In summary, this paper demonstrates that Phe in the NPLFY motif of the rat AT receptor is essential for normal agonist binding to the receptor, as well as for binding of the nonpeptide antagonist, losartan. These findings may explain the competitive kinetics of the binding of many nonpeptide Ang II antagonists, despite their additional dependence on other residues that are not required for peptide binding(39, 40) . Our data also support the predicted importance of Asn in the activation of G protein-coupled receptors, but are not consistent with the general importance of the conserved NPXY sequence in agonist-induced endocytosis of these receptors.

  
Table: Parameters of [I-Sar,Ile]Ang II binding for wild-type and mutant AT receptors expressed in COS-7 cells

The data are expressed as means ± S.E. of three independent experiments each performed in duplicate.


  
Table: IC of I-Ang II binding in the absence or presence of 5 µM GTPS and EC of the combined InsP + InsP responses for wild-type and mutant AT receptors expressed in COS-7 cells

The binding data are expressed as means ± S.E. of three independent experiments each performed in duplicate. The EC values are shown as means ± S.E. from independent dose-response curves of two to three experiments.



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.

§
Present address: Dept. of Physiology, Semmelweis University 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: AT receptor, type 1 angiotensin II receptor; Ang II, angiotensin II; InsP, inositol bisphosphate; InsP, inositol trisphosphate; GTPS, guanosine 5`-3-O-(thio)triphosphate.


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