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Molecular Endocrinology, doi:10.1210/me.2006-0018
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Molecular Endocrinology 20 (8): 1924-1934
Copyright © 2006 by The Endocrine Society

Identification of Structural Determinants for G Protein-Independent Activation of Mitogen-Activated Protein Kinases in the Seventh Transmembrane Domain of the Angiotensin II Type 1 Receptor

Daniel K. Yee, Aae Suzuki, Laiyi Luo and Steven J. Fluharty

Department of Animal Biology (D.K.Y., A.S., L.L., S.J.F.) and the Institute of Neurological Sciences (S.J.F.), University of Pennsylvania, Philadelphia, Pennsylvania 19104-6046

Address all correspondence and requests for reprints to: Daniel K. Yee, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, 222E, Philadelphia, Pennsylvania 19104-6046. E-mail: dkyee{at}vet.upenn.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although the intrareceptor mechanisms whereby the angiotensin II (AngII) type 1 receptor activates phospholipase C (PLC) have been extensively investigated, analogous studies of signaling through mitogen-activated protein kinases (MAPK) have been lacking. We investigated MAPK activation and traditional Gq/PLC signaling in transfected cells using AngII and the signaling selective agonist [Sar1,Ile4,Ile8] AngII (SII). SII stimulated MAPK without inositol trisphosphate (IP3) production and thereby stabilizes an activated receptor state linked to G protein-independent MAPK signaling. Using receptor mutagenesis, we focused on the seventh transmembrane domain and identified three key residues—Tyr292, Phe293, and Thr287. At least three distinct activated states were revealed: 1) an AngII-stabilized state linked to Gq/PLC signaling, 2) an AngII-stabilized state connected to G protein-independent MAPK activation, and 3) a SII-stabilized state associated with G protein-independent MAPK signaling. The mutant Y292F failed to exhibit AngII-induced IP3 turnover yet remained capable of AngII-induced MAPK activation. SII failed to stimulate MAPK in Y292F-transfected cells. Thus, Tyr292 is a key epitope for activated states 1 and 3 but not required for activated state 2. Although the F293L mutant retained normal AngII responses, it also showed an IP3 response to SII, indicating that Phe293 may be involved in constraining the receptor to its inactive state. Mutations of Thr287 abolished all SII-induced signaling without affecting any AngII responses. Thr287 therefore represents a key residue for a SII-stabilized activated state. Taken together, the data identified a novel structural requirement (Thr287) for the SII-stabilized activated state and redefined the mechanistic roles for Tyr292 and Phe293.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE OCTAPEPTIDE ANGIOTENSIN II (AngII) mediates the biological effects of the renin-angiotensin system, which include stimulation of salt appetite and thirst (1), vasoconstriction (2), increased secretion of aldosterone from the adrenal cortex (3), and proliferation of cardiac tissue (4) and vascular smooth muscle (5, 6). AngII induces its biological effects in target tissues by binding to and activating G protein-coupled receptors (GPCRs), of which there are two main subtypes, designated the type 1 (AT1) and the type 2 (AT2) AngII receptors (7, 8). Numerous studies have established that the major cardiovascular, renal, and behavioral effects classically associated with AngII are mediated primarily through its interaction with the AT1 receptor rather than the AT2 subtype (9, 10).

Like other GPCRs, agonist binding at the AT1 receptor initiates a host of intracellular events (for review, see Ref. 11). For example, this receptor is well known to couple to the G protein Gq, which stimulates phospholipase C (PLC) to generate the second messengers diacylglycerol and inositol trisphosphate (IP3). Diacylglycerol and IP3 then respectively activate protein kinase C (PKC) and liberate intracellular stores of calcium. In addition to this more traditionally ascribed signaling pathway, there is growing evidence that AT1 receptor activation stimulates intracellular events previously attributed primarily to growth factor receptors. The discovery that AT1 receptors can mediate these alternate signaling pathways, including regulation of tyrosine kinase and tyrosine phosphatase activity as well as increased expression of several early immediate genes including c-fos, c-jun, and c-myc (12, 13) provides cellular mechanisms whereby AngII can exert persistent physiological and behavioral changes. Among the specific, alternate signaling pathways engaged by the AT1 receptor is the phosphorylation (activation) of mitogen-activated protein kinase (MAPK) family members, including p44/42MAPK, also known as ERK1/ERK2 (14).

Although AngII-induced MAPK activation was shown to require activation of PKC (15), it is becoming increasingly clear that the AT1 receptor can also initiate MAPK signaling through alternate means. Many of the mutational substitutions of the receptor that impaired agonist-induced Gq/PLC/PKC signaling did not affect the ability of the receptor to activate MAPK (16, 17, 18). Moreover, specific receptor mutations known to impair G protein coupling and activation did not abolish agonist-induced MAPK activation (17, 19).

Strategies to understand the signaling properties of AngII receptors have also relied on analogs of AngII. One such analog is a double isoleucine substituted form, [Sar1,Ile4,Ile8] AngII (SII) (20). Previous studies used this peptide analog exclusively in vitro as part of an effort to demonstrate that the Tyr4 and Phe8 residues of AngII are important pharmacophores, each of which is critical for the peptide’s bioactivity (20). Not surprisingly, these early experiments focused primarily on the ability of AT1 to activate the PLC/PKC/IP3 pathway. More recent studies, however, demonstrated that when MAPK, rather than IP3 was measured, SII acted as an agonist at the AT1 receptor independent of G protein activation in vitro (19, 21, 22) and in vivo (23). Coupled with the mutagenesis data, the use of SII to functionally probe the AT1 receptor further supports the growing evidence that the traditionally ascribed G protein-dependent pathway and the more recently discovered MAPK stimulation can occur independently.

In contrast to investigation of AT1 receptor activation of the traditionally assayed effector PLC, study of the structural requirements of signaling through alternate pathways such as MAPK has been limited. Considering the emerging importance of AT1 receptor signaling through MAPK, especially with respect to the mitogenic actions of AngII on cardiovascular tissues, it seems particularly imperative to understand the structural requirements for receptor activation of this pathway in addition to the traditional Gq/PLC/PKC pathway. Because of the importance of the seventh transmembrane domain (TM7) in mechanisms that define the shift from inactive state to activated state linked to Gq/PLC/PKC signaling, we began our investigation of the receptor’s G protein-independent MAPK signaling at this critical receptor domain by comparing the structural determinants and mechanism for AT1 receptor activation of the more recently identified effector MAPK with those long-established for the activation of PLC and phosphoinositide hydrolysis. Using receptor mutagenesis, our experiments identified a novel structural requirement for the SII-stabilized receptor activation and redefined the mechanistic roles for other key TM7 residues in maintaining and stabilizing AT1 receptor activated states.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Conservative Segment Exchange (CSE) Mutation of TM7
To begin evaluating the involvement of TM7 in initiating both traditional Gq/PLC/PKC signaling and G protein-independent MAPK activation, we constructed a large-scale mutation of this domain. Because previous chimeric exchange of the AT1 TM7 with that of the AT2 receptor only partially blunted agonist-induced MAPK activation (18), we designed an even more disruptive mutational change by using CSE—an approach that substitutes entire segments of a protein with different, yet chemically similar residues. In designing the TM7 mutation, exceptions to substitution include amino acids known to play key structural roles (i.e. proline, glycine, and cysteine) as well as residues conserved in both AT1 and AT2 receptors and the NPXXY sequence at the bottom of TM7 because this structural motif is known to be important in receptor activation for numerous GPCRs (24). The resulting mutant, AT1 TM7 CSE, is shown in Fig. 1Go.


Figure 1
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  		Fig. 1. Comparison of Amino Acid Sequences of the TM7 of AT1 and AT2 Receptors with That of the AT1 TM7 CSE Mutant

With respect to TM7, comparison of the sequences of the AT1 and AT2 receptors with the CSE mutant is shown above. Based on IP3 assays, key epitopes previously reported for the AT1 receptor include Tyr292, Asn294, and to a lesser degree Asn295 (highlighted by gray shaded boxes); their substitutions impaired AngII-induced IP3 turnover. We targeted Thr287 and Phe293 (highlighted in black boxes) as candidate residues that may contribute toward receptor mechanisms linked with G protein-independent activation of MAPK.

 
When transfected into COS-1 cells, the CSE mutant was successfully expressed as demonstrated by binding assays using 125I-labeled [Sar1,Ile8] AngII in saturation isotherms (Table 1Go; KD = 3.42 ± 0.71 nM, BMAX = 30.3 ± 2.6 fmol/mg protein; n = 3). We then investigated the relative abilities of AngII and SII to activate two distinct signaling pathways—stimulation of the PLC/PKC/IP3 pathway and activation of MAPK. As shown in Fig. 2Go, treatment of AT1-transfected cells with 1 µM AngII led to reliable increases in both IP3 formation and in activation (i.e. phosphorylation) of MAPK. Cells treated with 30 µM SII also exhibited reliable increases in activated MAPK without IP3 formation. In comparison, the cells transfected with the AT1 TM7 CSE mutant failed to exhibit either IP3 production or MAPK activation when treated with either AngII or SII.


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  		Table 1. 125I-Labeled [Sar1,Ile8] AngII Binding Affinities of the Wild-Type and Mutant TM7 Receptors

 

Figure 2
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  		Fig. 2. Relative Efficacies of Wild-Type AT1 Receptor and AT1 TM7 CSE Mutant Receptor to Activate IP3 Production and MAPK in Transfected COS-1 Cells

COS-1 cells transfected with either wild-type or mutant AT1 TM7 CSE receptors were treated with either vehicle, 1 µM AngII, or 30 µM SII. A, Relative efficacies of the receptors to activate IP3 production in transfected cells. [3H]Inositol-loaded cells were treated with each drug for 30 sec. The cells were then lysed and analyzed for [3H]IP3. The values reported represent the mean percentage of vehicle levels ± SEM (n = 5). B, Relative efficacies of the receptors to activate MAPK. Transfected cells were serum-starved overnight and then treated with each drug for 5 min. Cell lysates were then subjected to SDS-PAGE followed by immunoblotting for phosphorylated (activated) MAPK. Normalized levels of phosphorylated (activated) MAPK were averaged (means ± SEM, n = 4) and graphed as a percentage of the mean vehicle-treated levels in AT1-transfected cells. A representative immunoblot for activated (phosphorylated) MAPK is shown below the graph. Similar immunoblots for total (i.e. phosphorylated and nonphosphorylated) MAPK indicated that total levels remained unchanged (data not shown). *, Significantly different from vehicle-treated levels (P < 0.01).

 
Point Mutations of TM7
Because whole-scale substitutions represented in the mutant AT1 TM7 CSE impaired SII- and AngII-induced MAPK activation, some of the residues that were changed may play a role in a partially activated state of the receptor capable of initiating G protein-independent cell signaling as indicated by MAPK activation without IP3 turnover. Therefore, we carefully compared the TM7 sequences of AT1 and AT2 receptors with the CSE mutant (Fig. 1Go) for candidate residues. The comparison of the three amino acid sequences yielded Thr287 and Phe293 (Fig. 1Go) as potential candidate residues that may contribute toward receptor activated state(s) resulting in MAPK activation in the absence of PLC/PKC/IP3 signaling. We subsequently constructed two point mutants for each position: conservative substitutions (T287S and F293Y) as well as nonconservative substitutions (T287V and F293L). In addition, the residue Tyr292 was also evaluated (via the mutant Y292F) for comparison.

When transfected into COS-1 cells, the mutants Y292F, T287S, T287V, and F293L were each highly expressed and all exhibited high affinity for 125I-labeled [Sar1,Ile8] AngII in saturation isotherms (Table 1Go). In contrast, membrane preparations from F293Y-transfected cells, failed to demonstrate any radioligand binding. The lack of binding activity by the F293Y mutant could suggest either very weak affinity for the radioligand that is undetectable by the equilibrium binding protocol or that this construct may not be expressed in our transfected cell system. It is important to note that the sister mutation of this phenylalanine to a leucine (F293L) was highly expressed when transfected in cells and efficiently bound radioligands (Table 1Go). Because the F293L mutation represents a less conservative substitution at this residue position than F293Y, the failure to detect any radioligand binding by F293Y suggested that this mutant receptor construct was not expressed in our transfected cell system. Despite the likelihood that F293Y was not being expressed when transfected into cells, we still included this mutant construct in our evaluation of every TM7 single point mutant in the functional assays.

Figure 3Go summarizes the functional responses of each of these mutant receptors with respect to IP3 production and MAPK activation under either AngII or SII treatment. Confirming previous studies that identified Tyr292 as a key structural position with respect to receptor activation (25, 26), the mutant Y292F was unable to exhibit AngII-induced IP3 turnover yet remained capable of AngII-induced MAPK activation. Extending the functional characterizations of the Y292F mutant with SII treatments suggested that the substitution also impaired G protein-independent MAPK activation by this signaling selective agonist; the SII-induced MAPK activation observed for the wild-type receptor was absent for Y292F. The two mutations of Thr287 resulted in similar functional profiles. With respect to AngII treatment, both T287S and T287V responded with increased IP3 production and activation of MAPK. The two mutants, however, failed to exhibit the SII-induced MAPK signaling found with the wild-type receptor. The substitutions of Phe293 yielded differing observations. F293Y-transfected cells failed to demonstrate any agonist-induced functional changes. Coupled with the aforementioned failure to detect any ligand binding activity, these data suggest that this mutant receptor was not expressed when transfected into COS-1 cells. F293L, in contrast, is highly expressed and also functionally responded to both AngII and SII. AngII treatment resulted in IP3 activation and MAPK stimulation. SII also induced MAPK activation for this mutant receptor. Surprisingly, SII, which acted as an antagonist for wild-type AT1-mediated IP3 production (Fig. 2Go) (23), showed some agonist activity for F293L with respect to this G protein-mediated signaling pathway (Fig. 3Go). To more fully characterize SII’s agonist activity on F293L, dose-response curves for cells transfected with F293L were conducted in response to AngII and SII (Fig. 4Go). As expected, AngII acted as a full agonist for the AT1 F293L receptor (significant increases in IP3 production were observed by 10–8 M, P < 0.01; EC50 = 3.18 ± 0.07 nM). The mutation of Phe293, however, also now permitted SII to act as a partial agonist (significant levels of IP3 production by 10–6 M, P < 0.01; EC50 = 446 ± 120 nM).


Figure 3
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  		Fig. 3. Relative Efficacies of Wild-Type AT1 and TM7 Point Mutant Receptors to Activate IP3 Turnover and MAPK in Transfected Cells

Transfected cells were treated with either vehicle, 1 µM AngII, or 30 µM SII as outlined in Fig. 2Go. A, Relative efficacies of receptors to activate IP3 production in transfected cells. The values reported represent the mean ± SEM (n = 3–5). B, Relative efficacies of the receptors to activate MAPK. Normalized levels of activated MAPK for each receptor were averaged (means ± SEM, n = 4) and graphed as a percentage of the mean levels in vehicle-treated AT1-transfected cells. Representative immunoblots for activated (phosphorylated) MAPK are shown below the graph. *, Significantly different from vehicle-treated levels of AT1-transfected cells (P < 0.01). **, Significantly different from vehicle-treated levels of AT1-transfected cells (P < 0.05).

 

Figure 4
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  		Fig. 4. Dose-Response Curves of the Mutant Receptor AT1 F293L to Activate IP3 Production in Response to AngII and SII

AT1 F293L-transfected cells were metabolically labeled with [3H]inositol and treated with increasing concentrations of either AngII (from 10–12–10–6 M) or SII (from 10–10–10–4 M) for 30 sec. The values reported are the means ± SEM (n = 3). *, Significantly different from vehicle-treated levels of transfected cells (P < 0.01).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Because of its long-standing association with the AT1 receptor, stimulation of PLC and the resultant release of IP3 from membrane phospholipids has been the commonly used index for detecting and measuring the activated state of the AT1 receptor. More specifically, studies have identified those amino acids participating in and controlling the agonist-dependent conformational change of the AT1 receptor from the inactive (nonsignaling) to the activated (promoting signaling) state as defined by the Gq/PLC/PKC pathway. Many of these residues reside in transmembrane-spanning domains. For example, key residues identified by mutagenesis include Asp74 in TM2 (27) and Asn111 in TM3 (28, 29, 30), as well as Tyr292 (25), Asn294 (31, 32), and Asn295 (26, 33) in TM7. Amino acid substitutions at these residues either significantly inhibit or otherwise abolish the ability of AngII to stimulate IP3 release without affecting the affinity of the receptor for AngII or G protein coupling. Indeed, identification of the key AT1 residues also point to common receptor activation mechanisms among GPCRs in general. For example, Asp74 is both widely conserved among GPCRs and equally crucial for their agonist-dependent signaling (34, 35, 36).

Further demonstration in the involvement of the transmembrane-spanning domains has been shown through studies that chemically probe the water accessibility of these domains within the AT1 receptor. Based on the use of the substituted cysteine accessibility method (SCAM) and of photoaffinity probes, the ligand binding pocket of the receptor is defined, in part, by residues within TMs 3, 6, and 7 (31, 32, 37, 38). Coupled with analysis of the constitutively active AT1 N111G mutant receptor, these approaches have suggested a specific rotation of TM3 as well as an outward translation of TM7 upon activation of the receptor (37, 38). Because of the convergence of mutational, SCAM, and photoaffinity probe data, the involvement of these TMs with respect to agonist-induced IP3 turnover is firmly established, with several models proposed to describe interactions between the transmembrane domains upon AT1 receptor activation (31, 32, 37, 38, 39, 40).

Evidence for multiple activated states of the AT1 receptor was initially suggested by data revealing that the structural requirements for AT1 receptor activation defined by assays of IP3 levels may not be applicable when defining receptor activation by monitoring MAPK signaling. For example, substitution of the conserved TM2 aspartate (Asp74) and key TM7 residues (Tyr292 and Asn295) blocked AngII-induced IP3 without affecting MAPK signaling (16, 18). Other studies have further indicated that AngII-induced MAPK activation can even proceed without G protein coupling and activation (17, 19). Moreover, the AngII analog SII has been shown to initiate AT1-mediated MAPK activation in the absence of IP3 turnover (19, 21, 22, 23). Thus, the AT1 receptor is clearly capable of maintaining multiple activated states, some of which may be linked to G protein-independent signaling.

Because a previously reported chimeric receptor (AT1[AT2 TM7]) exchanging the AT1 receptor TM7 with that of the AT2 receptor partially blunted AngII-induced MAPK activation (18), we focused our investigation on the AT1 receptor’s TM7 in search of structural elements that may be linked to G protein-independent signaling. We sought a more complete inhibition of this functional response and made use of CSE. Despite the large-scale changes, radioligand binding assays demonstrated that the resulting AT1 TM7 CSE mutant (Fig. 1Go) was expressed when transfected into cells, albeit at a much lower level compared with wild-type receptor (Table 1Go). The transfected cell system used in the present study typically overexpresses transfected receptors as indicated by the very high expression levels (BMAX) of the AT1 receptor as well as many of the mutant constructs (Table 1Go). Despite its relatively lower level of expression of the AT1 TM7 CSE mutant, in our experience, the expression level of the mutant receptor was sufficient for its functional characterization. Indeed, in a previous report, we functionally characterized an AT1/AT2 chimeric receptor whose expression was comparable to AT1 TM7 CSE; cells transfected with the AT1/AT2 chimeric receptor responded to both AngII and the AT2-specific agonist CGP 42112A with increased IP3 production comparable to the wild-type AT1 receptor (41). With respect to the present study, functional characterizations of AT1 TM7 CSE showed that the large segment substitution abolished any functional response to either AngII or SII treatment (Fig. 2Go). Although the effect of such a large-scale mutation on agonist-induced IP3 production was not surprising, the additional inhibition of MAPK activation was a first step in suggesting any involvement of TM7 toward an AT1 receptor activation mechanism that is independent of G protein activation and linked to MAPK signaling.

The functional characterization of the AT1 TM7 CSE mutant indicated a role, either directly or indirectly, of some of the substituted residues in an activated receptor state identified by G protein-independent cell signaling. Coupled with the partial blockade of AngII-induced MAPK by the previously reported AT1[AT2 TM7] chimera, we therefore compared the TM7 sequences of AT1 and AT2 receptors with that of the CSE mutant (Fig. 1Go) for candidate residues to be targeted in further mutational analysis. Previously, it was shown that Tyr292 and to a lesser degree Asn295 are key epitopes for IP3-defined AT1 receptor activation. Because their substitutions impaired AngII-induced IP3 turnover but not MAPK activation (16, 17, 18), these two positions were excluded as candidates. Asn294, which is also conserved in the AT2 receptor, has been shown to be a key site of interaction with AngII and its analogs (31, 32). Due to this putative role linking AngII to the receptor, this position was also removed from the target list. Additionally, residues that may play simply a steric, place-holding role were also eliminated as candidates. Although residues in those positions when substituted could impair receptor activation, because of the chemical nature of their side chains (e.g. leucines, isoleucines, alanines, and valines), such substitutions were more likely to induce indirect structural perturbations to TM7 rather than interacting directly with receptor activation mechanisms. Consequently, with these issues in mind, Thr287 and Phe293 were targeted as potential candidates. The subsequent mutations of these two positions were also compared with Tyr292 (via the mutant Y292F, a substitution to the AT2 homolog). Figure 5Go shows the relative positions of these residues within TM7 with the relative rotational orientation of TM7 as defined by the SCAM study of Boucard et al. (37).


Figure 5
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  		Fig. 5. Relative Positions of Targeted TM7 Residues in the Present Study

The relative positions of Thr287, Tyr292, and Phe293 within TM7 and to other transmembrane domains in the AT1 receptor are highlighted in the helical wheel shown. The relative orientation of the TM7 is aligned to the other TM domains based on SCAM data by Boucard et al. (37 ).

 
As shown in Fig. 3Go and confirming previous studies that identified Tyr292 as a key residue with respect to AT1 receptor activation (25, 26), its substitution to the AT2 receptor counterpart phenylalanine rendered the mutant receptor incapable of initiating AngII-induced IP3 turnover. With respect to MAPK signaling and in agreement with previous data (18), the Y292F mutant continued to demonstrate AngII-induced MAPK activation. Extending the MAPK characterizations with SII treatments showed that Tyr292 may be a structural requirement of SII-stabilized receptor activation as well; the SII-induced MAPK activation observed for the wild-type receptor was absent for Y292F. Based on these data, there are at least three distinct activated receptor states: 1) an AngII-stabilized state linked to Gq/PLC/PKC signaling, 2) an AngII-stabilized state connected to G protein-independent MAPK activation, and 3) a SII-stabilized state associated with G protein-independent MAPK signaling.

The exact nature that Tyr292 participates in the receptor mechanism(s) defined by AngII-induced IP3 production and SII-induced MAPK activation remains unclear. Based on the well-established impairment of AngII-induced IP3 production by mutations of Tyr292 and Asn295, receptor activation mechanisms have been proposed that highlight a direct involvement of Tyr292 within the receptor states, including a direct linkage between Tyr292 and Asn111 in the inactive ground state of the receptor with a shift to a hydrogen bond between Tyr292 and Asp74 upon receptor activation (39). Moreover, because Tyr292 was substituted to its AT2 receptor counterpart phenylalanine, it could be argued that the proposed activation mechanisms were unique to the AT1 subtype. However, we have previously shown that complete substitution of the segment encompassing AT1’s TM7 to its cytoplasmic tail with the reciprocal AT2 portion resulted in a chimeric receptor that exhibited AT1 wild-type AngII-induced IP3 production, despite the fact that this chimeric exchange substituted both Tyr292 and Asn295 (26). Furthermore, a simultaneous substitution of both positions to their AT2 counterparts (i.e. Y292F/N295S) yielded a mutant receptor that retained its wild-type AngII-induced IP3 response (26). These observations suggest that, if any linkages exist between Tyr292 and other AT1 residues, they would not be via hydrogen bonds. Moreover, a direct participatory role by Tyr292 upon AT1 receptor activation is less likely.

Alternatively, it is possible that the positions occupied by AT1’s Tyr292 and Asn295 and by their AT2 counterparts (respectively, Phe308 and Ser311) represent within the TM7 domains of both subtypes key structural points, where single substitutions can introduce changes in the overall conformation of both TM7s. The positions may represent uniquely paired residues for each subtype and any subtle shift to the overall conformation of TM7 by one residue is match to a compensating countershift of the other residue. Thus, although the TM7s of both subtypes may adopt similar overall conformational profiles, a single substitution at either of these two positions (AT1’s Tyr292 and Asn295; AT2’s Phe308 and Ser311) would introduce a kink that results in misalignment of TM7 and thereby would impair the overall function of TM7. The impact of the Y292F substitution on receptor function may therefore be the result of indirect conformational perturbations from the mutation rather than disruption of a direct link between Tyr292 and another key residue(s). The double mutation Y292F/N295S restored wild-type IP3 responses to AngII in the receptor, because the simultaneous N292F substitution corrected the original Y292F kink that had initially skewed TM7’s conformation and alignment. The Y292F mutation also impaired SII-induced MAPK activation and therefore is consistent with an indirect role of Tyr292 with respect to receptor activation by the signaling selective agonist. Still, abolishing SII-induced MAPK activation confirmed the involvement of TM7 with respect to its overall conformation and alignment for an activated receptor state linked to SII binding that was first suggested by the AT1 TM7 CSE mutant.

Phe293 was another TM7 residue that was targeted in our mutational analysis. We made two substitutions—one conservative (to tyrosine) and another nonconservative (to leucine). Whereas the F293Y mutant was not expressed in transfected cells, the F293L construct was highly expressed, efficiently bound radioligands (Table 1Go), and was amenable to functional characterizations. Previous studies pairing photolinking AngII analogs with selective methionine substitutions within AT1’s TM7 have indicated that the C-terminal residue of AngII and its analogs intersect deeply into the receptor’s TM7, specifically in the vicinity of Phe293 and Asn294 (31, 32). Subsequent functional evaluations of F293M and N294M revealed differences in their impact of AngII-induced IP3 production; the N294M mutant displayed impaired agonist-induced IP3 turnover, whereas the F293M mutant did not. Consistent with these previous reports, our F293L mutation did not block AngII-induced IP3 production. Not surprisingly, because Gq/PLC/PKC signaling can subsequently induce MAPK activation (15), AngII also stimulated MAPK activation for the F293L mutant. We then extended our functional analysis with the AngII analog SII. In transfected cells, as shown here and in several reports, whereas SII is bound by wild-type AT1 receptors and induces MAPK activation, it fails to stimulate IP3 production (19, 21, 22). Thus, although SII is an AT1 receptor agonist based on MAPK activation, this ligand is an AT1 receptor antagonist with respect to IP3 turnover. Indeed, we have previously demonstrated that SII blocked AngII-induced IP3 production in AT1-transfected cells and have also extended these observations in vivo (23). Yet, SII acted on F293L as a partial agonist for both IP3 and MAPK signaling (Figs. 3Go and 4Go). Because the studies using photoaffinity probes revealed that the C-terminal residue of AngII and its analogs may intersect in the vicinity of both Phe293 and Asn294, it is possible that our substitution of Phe293 now permitted a novel, direct association of SII’s C-terminal isoleucine with this TM7 position. However, the lack of any functional consequence with AngII treatment for two different mutations at this position, our F293L and the previously reported F293M, would suggest that, unlike the other photolinked position Asn294, Phe293 may not directly interact with agonists despite being capable of labeling by photoaffinity probes. Consequently, it is less probable that our F293L mutation created a new opportunity for SII and receptor to directly interact. Instead, Phe293 may be involved in maintenance of the inactive ground state of the receptor and that its substitution resulted in a receptor that was more permissive toward an activated receptor state identified by increased IP3 production. We have previously shown that complete substitution of the segment encompassing AT1’s TM7 to its cytoplasmic tail with the reciprocal AT2 segment resulted in a chimeric receptor that demonstrated greater laxity in that peptidic antagonists such as [Sar1,Ile8] AngII acted as full agonists (26). The chimeric exchange may have disrupted AT1-specific residues that maintain the receptor subtype in its inactive state. Indeed, AT1’s Phe293 is not conserved in the AT2 subtype. Miura and Karnik (20) have also demonstrated that the AT1 and AT2 subtypes interact with AngII and its analogs differently, with the AT1 receptor being more constrained and the AT2 subtype being more relaxed. Phe293 may therefore play a part in constraining the AT1 receptor in its inactive ground state, with the shift toward receptor activation disrupting the association of Phe293 to another as-yet-unidentified residue(s). If true, the F293L substitution weakened a constraint on the inactive ground state of the receptor and thereby rendered the receptor more permissive toward activation. That the single F293L mutation did not generate a constitutively active receptor would indicate that multiple linkages are involved in constraining the AT1 receptor in its inactive state.

Another TM7 residue that we targeted was Thr287. As shown in Fig. 3Go, both the conservative (T287S) and nonconservative (T287V) substitutions of this residue did not alter receptor response to AngII, exhibiting both increased IP3 production and MAPK activation. Because the Thr287 mutations did not affect AngII-driven functional responses, this residue is not involved in either direct interaction with AngII or with the receptor activation mechanisms initiated upon binding with AngII. In contrast, the mutations did alter receptor response to SII. Whereas the wild-type receptor responded to SII treatment with MAPK activation, the peptide failed to induce either T287S or T287V to signal through this pathway. Thus, Thr287 is identified as a unique structural requirement for an activated receptor state stabilized by SII. Combined with the results of the Y292F mutant, the Thr287 mutants revealed that there are differences in the manner that AngII and SII interact with the receptor.

The present study characterized the effects of mutations on three TM7 residues (Thr287, Tyr292, and Phe293) of the AT1 receptor with respect to two functional responses—IP3 production and MAPK activation. Based on our experiments, we report on evidence of a unique structural requirement (Thr287) for the SII-stabilized activated receptor state that is linked to G protein-independent MAPK activation. Moreover, our data suggest revised roles for two other key TM7 residues, Tyr292 and Phe293, as well provide evidence of at least three distinct activated states for the receptor. A schematic diagram shown in Fig. 6Go summarizes the inactive state in relation to the proposed activated states and their structural requirements based on the presented data. In the study of receptor activation mechanisms, the activated state linked to AngII-induced IP3 production has been the best defined and has led to proposed mechanisms for the shift from inactive receptor to an activated state associated to the Gq/PLC/PKC pathway (37, 38, 39, 40). The mechanisms involved in receptor activated states linked to G protein-independent signaling, e.g. SII-induced or AngII-induced MAPK activation in the absence Gq/PLC/PKC signaling, are much less understood and must be accounted within the better studied AngII-stabilized state associated with IP3 production. Based on SCAM studies of wild-type AT1 receptor and the constitutively active mutant N111G, it has been postulated that, upon binding with AngII, several important transmembrane movements occur. Of particular interest, there is a slight counterclockwise rotation of TM3 and a translational movement away from the ligand binding pocket of TM7 (37, 38). Using the present mutational data, it is possible that the two activated states tied to G protein-independent MAPK activation are stabilized before the full outward translation of TM7 induced upon AngII binding.


Figure 6
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  		Fig. 6. A Schematic Diagram Indicating the Presence of Multiple Activated States for the AT1 Receptor

The schematic diagram indicates multiple activated states for the AT1 receptor. The structural requirements revealed by the TM7 point mutants are shown.

 
With the discovery that GPCRs can mediate signaling through MAPK, there has been extensive investigation of possible cellular mechanisms that connect activated GPCRs to this signaling pathway. One mechanism is through transactivation of receptor tyrosine kinases, such as the epidermal growth factor (EGF) receptor (42). Indeed, the AT1 receptor has been linked to the EGF receptor transactivation, although the exact link between the two receptor systems remains an active area of research. One possible cellular mechanism involves specific metalloproteases as the target of PKC activation. Stimulation of the AT1 receptor would then lead to the release of preformed EGF receptor ligands, which then stimulate the EGF receptor and activate the MAPK cascade (43, 44). EGF transactivation may also arise from functional complexes between AT1 receptors and EGF receptors. Seta and Sadoshima (45) have shown that the AT1 cytoplasmic tail is involved in the formation of these receptor complexes as a mutation of Tyr319 within the highly conserved YIPP motif, which is a linkage point to several signaling molecules, blocked EGF receptor transactivation. In addition, the AT1 receptor-induced signaling prerequisites for EGF receptor transactivation remain an open question. Although there is some evidence that Ca2+ and perhaps Gq/PLC/PKC signaling are necessary (44), Miura et al. (22) have reported that SII, which does not stimulate IP3 production, is still able to initiate AT1 receptor transactivation of EGF receptors in human coronary artery smooth muscle cells. Thus, the activated AT1 receptor states that stimulate MAPK signaling in the absence of Gq/PLC/PKC signaling, i.e. associated with either SII binding or revealed by AngII binding to the AT1 Y292F receptor mutant, may not be activating MAPK through EGF receptor transactivation.

GPCR signaling through the MAPK cascade may also be activated through interactions with ß-arrestin (42). Studies of the ß2 adrenergic receptor have shown that ß-arrestin-Src complexes mediate activation of the MAPK cascade (46) and may also act as a scaffold to bring together members of the MAPK cascade (47). Indeed, not only has the link between the AT1 receptor, ß-arrestin, and MAPK signaling been established, it has also been shown that this cellular mechanism may be independent of Gq/PLC/PKC signaling (19). Therefore, ß-arrestin may also serve as the connector between the activated states stabilized by SII or revealed by AngII binding to the AT1 Y292F receptor mutant and subsequent MAPK signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
AngII was purchased from Bachem (King of Prussia, PA). SII was synthesized and obtained from Bachem. Tissue culture medium and supplements, including LipofectAMINE reagent were obtained from Invitrogen Life Technologies (Gaithersburg, MD). [3H]Myoinositol was obtained from American Radiolabeled Chemicals (St. Louis, MO) and 125I-labeled [Sar1,Ile8] AngII was obtained from the University of Mississippi Peptide Center, University, MS (directed by Dr. Robert Speth). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted.

Mutagenesis
A modified version of the splicing by overlap extension (SOE) technique was used to generate the AT1 receptor mutants (48). This procedure involved two steps: 1) introduction of the desired base substitution into the AT1 cDNA receptor using specifically designed complementary and overlapping primers, followed by 2) amplification of the mutated cDNA using PCR. As a refinement to enhance the fidelity of SOE, a small amount of Pfu DNA polymerase (1:100 Pfu:Taq) was added. The primers used were as follows: AT1 TM7 CSE, 5'-GTGTTTTATAACCAGTGCATCAACCCTCTGTTTTACGGCTTTC-3' (forward/sense primer) and 5'-GATGCACTGGTTATAAAACACTAGGCAGATAGATAGAGGTAGGGCAGTGTCCACGATGTCGG-3' (reverse/antisense primer); AT1 Y292F, 5'-AGCGTTTTTTAACAACTGCCTGAACCC-3' (forward/sense primer) and 5'-GGCAGTTGTTAAAAAACGCTATGCAGATGGTTATGGG-3' (reverse/antisense primer); and AT1 T287S, 5'-CCATGCCCATATCCATCTGCATAGCGTATTTTAAC-3' (forward/sense primer) and 5'-CTATGCAGATGGATATGGGCATGGCAGTGTCCAC-3' (reverse/antisense primer); AT1 T287V, 5'-CCATGCCCATAGTCATCTGCATAGCGTATTTTAAC-3' (forward/sense primer) and 5'-CTATGCAGATGACTATGGGCATGGCAGTGTCCAC-3' (reverse/antisense primer); AT1 F293Y, 5'-CATAGCGTATTATAACAACTGCCTGAACCCTCTG-3' (forward/sense primer) and 5'-GGCAGTTGTTATAATACGCTATGCAGATGGTTAT-3' (reverse/antisense primer); and AT1 F293L, 5'-CATAGCGTATTTGAACAACTGCCTGAACCCTCTG-3' (forward/sense primer) and 5'-GGCAGTTGTTCAAATACGCTATGCAGATGGTTAT-3' (reverse/antisense primer). The first fragment was generated using the primers T7 and a reverse/antisense primer, whereas the second fragment was produced using primers SP6 and a forward/sense primer. Wild-type AT1 and AT2 cDNA served as the template in these PCRs for all of the mutants generated. Reaction conditions were 30 cycles of 94 C (1 min), 58 C (1 min), and 72 C (1 min). After purification using the Wizard PCR Preps DNA Purification System (Promega, Madison, WI), the two fragments were combined in the overlap extension reaction using the same PCR conditions as described. After production of the full-length chimeric receptor using SOE, the chimera was subcloned into the expression vector pCR3 (Invitrogen Life Technologies) and sequenced to confirm its validity.

Cell Culture Experiments
COS-1 cells were used for all of the in vitro experiments described here. Cells were grown in polystyrene tissue culture flasks in medium consisting of DMEM supplemented with 10% fetal bovine serum, L-glutamine, and penicillin-streptomycin in a humidified atmosphere of 5% CO2 and 95% O2 at 37 C. AT1 receptor was later introduced into the cells by transfection with LipofectAMINE for 5 h, after which the transfection medium was removed and replaced with normal growth medium. Transfected cells were used for ligand binding experiments, IP3 assays, and MAPK assays as described below.

Ligand Binding Experiments
Two days after transfection, the cells were rinsed with ice-cold Tris-buffered saline [20 mM Tris-HCl (pH 7.4), and 150 mM NaCl] and then harvested by scraping into a 20 mM Tris-HCl (pH 7.4). After polytron homogenization and centrifugation, the membrane pellets were resuspended in assay buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM MgCl2, 0.3 trypsin inhibitor units/ml aprotinin, and 100 µg/ml 1,10-phenanthroline] and protein content was determined by bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, IL). The binding assays were initiated by addition of the desired amount of 5 µg membrane protein to assay mixture containing various concentrations of 125I-labeled [Sar1,Ile8] AngII and unlabeled competitors (AngII and SII). Nonspecific binding was defined as the amount of radioligand binding remaining in the presence of 1 µM [Sar1,Ile8] AngII. The binding assays proceeded for 60 min at room temperature and were terminated by rapid dilution with 5 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1.5 mM CaCl2, and vacuum filtration on glass-fiber filters presoaked with 0.3% polyethylenimine, using a Brandell harvester (Brandell, Gaithersburg, MD). Radioligand binding was quantified by {gamma} counting of the filters. Data were analyzed and fit to a single-site model.

IP3 Assays
IP3 formation was measured as described previously (18). Briefly, AT1-transfected cells were preloaded with [3H]myoinositol (4.5 µCi/ml DMEM) for 18 h before being treated with vehicle (DMEM), AngII, SII, or both AngII and SII for 30 sec. Cells then were washed, lysed, and processed for IP3 quantification by stepwise elution from anion exchange resin columns. IP3 was quantified by liquid scintillation counting of the IP3-containing fraction. Protein quantification was performed by BCA assay.

In Vitro MAPK Assays
Activated p44/42MAPK was measured as described previously (49). Briefly, AT1-transfected cells were treated with vehicle (DMEM), AngII, or SII for 5 min before being washed, lysed by Dounce homogenization in cold lysis buffer [25 mM Tris-HCl (pH 8.0), 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM sodium molybdate, 1 mM phenylarsine oxide, 10 µg/ml pepstatin, 2 µg/ml aprotinin, and 10 µg/ml leupeptin], and processed for MAPK immunoblotting (described below) and protein quantification by BCA assay.

MAPK Immunoblotting
Western blotting for activated (phosphorylated) MAPK was performed as described previously (18). Briefly, 20–30 µg of protein was run on a 10% SDS-PAGE gel and subsequently transferred to a nitrocellulose membrane. Membranes were probed for activated MAPK by incubation with a monoclonal antibody directed against phospho-p44/42MAPK (1:1000; Cell Signaling Technology, Beverly, MA). The membranes then were washed and incubated with peroxidase-conjugated goat antimouse IgG (1:1000 or 1:2000; Jackson ImmunoResearch Laboratories, West Grove, PA) before immunoreactivity was detected using chemiluminescence reactions according to manufacturer’s instructions (Western Lightning kit; PerkinElmer Life Sciences, Boston, MA).

Data Analysis
Radioligand binding data were processed using Prism (version 4.0; GraphPad Software, San Diego, CA) to calculate Bmax and Kd values for 125I-labeled [Sar1,Ile8] AngII. Films from MAPK immunoblots were digitized, and the optical density of each band was determined using Scion Image (version 4.0.2; Scion Corp., Frederick, MD). Statistical comparisons using ANOVA were conducted with Prism (version 4.0; GraphPad Software).


   ACKNOWLEDGMENTS
 
We thank Dr. Derek Daniels for helpful comments during the preparation of this manuscript.


   FOOTNOTES
 
This work was supported by the following awards from the National Institutes of Health: HL058792 (to D.K.Y.) and DK052018 (to S.J.F.).

D.K.Y., A.S., L.L., and S.J.F. have no conflicts of interest to declare.

First Published Online March 23, 2006

Abbreviations: AngII, Angiotensin II; AT1, angiotensin II type 1 receptor; AT2, angiotensin II type 2 receptor; BCA, bicinchoninic acid; CSE, conservative segment exchange; EGF, epidermal growth factor; GPCR, G protein-coupled receptor; IP3, inositol trisphosphate; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; PLC, phospholipase C; SCAM, substituted cysteine accessibility method; SII, [Sar1,Ile4,Ile8] AngII; SOE, splicing by overlap extension; TM7, seventh transmembrane domain.

Received for publication January 10, 2006. Accepted for publication March 16, 2006.


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