Agonist-induced Phosphorylation of the Angiotensin II (AT1A) Receptor Requires Generation of a Conformation That Is Distinct from the Inositol Phosphate-signaling State*

G protein-coupled receptors are thought to isomerize between distinct inactive and active conformations, an idea supported by receptor mutations that induce constitutive (agonist-independent) activation. The agonist-promoted active state initiates signaling and, presumably, is then phosphorylated and internalized to terminate the signal. In this study, we examined the phosphorylation and internalization of wild type and constitutively active mutants (N111A and N111G) of the type 1 (AT1A) angiotensin II receptor. Cells expressing these receptors were stimulated with angiotensin II (AngII) and [Sar1,Ile4,Ile8]AngII, an analog that only activates signaling through the constitutive receptors. Wild type AT1A receptors displayed a basal level of phosphorylation, which was stimulated by AngII. Unexpectedly, the constitutively active AT1A receptors did not exhibit an increase in basal phosphorylation nor was phosphorylation enhanced by AngII stimulation. Phosphorylation of the constitutively active receptors was unaffected by pretreatment with the non-peptide AT1 receptor inverse agonist, EXP3174, and was not stimulated by the selective ligand, [Sar1,Ile4,Ile8]AngII. Paradoxically, [Sar1,Ile4,Ile8]AngII produced a robust (∼85% of AngII), dose-dependent phosphorylation of the wild type AT1A receptor at sites in the carboxyl terminus similar to those phosphorylated by AngII. Moreover, internalization of both wild type and constitutive receptors was induced by AngII, but not [Sar1,Ile4,Ile8]AngII, providing a differentiation between the phosphorylated and internalized states. These data suggest that the AT1A receptor can attain a conformation for phosphorylation without going through the conformation required for inositol phosphate signaling and provide evidence for a transition of the receptor through multiple states, each associated with separate stages of receptor activation and regulation. Separate transition states may be a common paradigm for G protein-coupled receptors.

Seven transmembrane-spanning receptors that couple to heterotrimeric guanyl nucleotide-binding proteins (G proteins) 1 are activated by an array of sensory and hormonal stimuli. Current theories (1)(2)(3)(4) for G protein-coupled receptor (GPCR) activation predict that receptors spontaneously isomerize between an inactive (R) and active (R*) state and that, in the absence of agonist, structural constraints maintain an equilibrium between R and R* that favors the inactive R state. The active R* conformation is selected (or induced) by agonist binding and couples to and activates G proteins, which initiate signaling. This two-state model has been revised to include two active states, R* and R**, in the so-called three-state model (5,6), which accommodates experimental evidence that one receptor can couple to different G protein effector pathways with distinct agonist potency profiles. In either model, the active states are then targeted for phosphorylation by specific GPCR kinases (GRKs), which only recognize the active conformation, and by second messenger-activated kinases (e.g. protein kinase C). Proteins termed arrestins bind to phosphorylated receptors and sterically hinder further association of the receptor with G protein and thereby terminate signaling (7). For some GPCRs, arrestins also act as adaptors to target the receptors for clathrin-mediated internalization (7) and to promote coupling to tyrosine kinase signaling pathways (8).
Experimental evidence for R and R*/R** has come from the observation that overexpression of many GPCRs leads to some degree of constitutive (or agonist-independent) activity, ostensibly by increasing the overall amount (not the proportion) of R*/R** available to interact with G proteins (1,4). Moreover, constitutively active GPCRs can arise from naturally occurring and engineered mutations (9), presumably as a result of transforming the receptor to an active state. Generally, these constitutively active GPCRs have proven to be constitutively phosphorylated and desensitized (4), providing support for the model that the active state is also the conformation targeted for phosphorylation, internalization and desensitization.
Type 1 angiotensin receptors (AT 1 ) are GPCRs that mediate the actions of angiotensin II (AngII), an octapeptide hormone (Asp 1 -Arg 2 -Val 3 -Tyr 4 -Ile 5 -His 6 -Pro 7 -Phe 8 ) which regulates blood pressure and water and salt balance. Stimulation of AT 1 receptors (two subtypes, AT 1A and AT 1B , exist in rodents) leads to G␣ q/11 -mediated activation of phospholipase C-␤ 1 , which generates diacylglycerol and inositol (1,4,5)trisphosphate. No naturally occurring, constitutively active AT 1 receptor mutants have been reported, but engineered mutation of Asn 111 to glycine (N111G) or alanine (N111A), in the third transmembrane helix of the AT 1A receptor, leads to partial activation (10 -12). Thus, in the absence of AngII binding, the N111G mutant displays ϳ50%, and the N111A mutant ϳ20% (10), of the maximal agonist-induced production of inositol phosphates. In the wild type AT 1A receptor, interaction of Tyr 4 of AngII with Asn 111 in the receptor appears to act as the trigger to convert R to R* and allow receptor activation. Small side-chain substitutions (glycine/alanine) of Asn 111 in AT 1A presumably release this conformational switch, allowing constitutive activity and removing the requirement of Tyr 4 in AngII for maximal receptor activation.
In this study, we examined the phosphorylation and internalization of the N111A and N111G constitutively active mutants of the AT 1A receptor. We anticipated that these mutants would display an elevated basal phosphorylation to parallel their enhanced G protein-phospholipase C-␤ 1 coupling. Instead, we observed that the phosphorylation of the constitutively active AT 1A receptors is not increased basally or following stimulation by AngII. The constitutively active receptors retained the ability to internalize in response to AngII. Rather surprisingly, the non-signaling AngII analog, [Sar 1 ,Ile 4 ,Ile 8 ]AngII, induced robust phosphorylation of the wild type AT 1A receptor, but this analog did not stimulate internalization of either wild type or constitutively active mutant AT 1A receptors. Thus, we provide evidence for the existence of multiple AT 1A receptor states capable of selectively mediating various aspects of receptor function.

EXPERIMENTAL PROCEDURES
Materials-The monoclonal antibody 1D4, produced by the Cell Culture Center (Endotronics Inc., Minneapolis), was a kind gift from Dr. Robert Graham (Victor Chang Cardiac Research Institute, Sydney, Australia); the 12CA5 monoclonal antibody was purified from hybridoma culture media using an influenza hemagglutinin antigen (HA)peptide affinity column; 125 I-AngII was provided by Dr. Conrad Sernia (Department of Physiology and Pharmacology, University of Queensland, Brisbane, Australia) or purchased from NEN Life Science Products at a specific activity of ϳ2000 Ci/mmol; EXP3174 was from the DuPont Merck Pharmaceutical Co. (Wilmington, DE); AngII from Auspep (Melbourne, Australia); [Sar 1 ,Ile 4 ,Ile 8 ]AngII from the Biotechnology Core Facility (Lerner Research Institute, Cleveland Clinic). Chinese hamster ovary cells (CHO-K1) were obtained from the American Type Culture Collection; The ExSite Mutagenesis kit was purchased from Stratagene; DNA modifying enzymes were from Promega; [ 32 P]orthophosphate were from Geneworks (Thebarton, Australia). ␣-Minimal essential medium, Opti-MEM, Hanks' buffered salt solution, and LipofectAMINE were purchased from Life Technologies, Inc., and protein A-agarose was from Roche Molecular Biochemicals. All other chemicals were from Sigma or BDH Laboratory Supplies.
Receptor Constructs and Expression-A synthetic AT 1A receptor gene, in the vector pMT-2, with the 1D4 epitope tag (TETSQVAPA) at the carboxyl terminus to allow immunoprecipitation and detection by Western blotting has been described earlier (10). Mutated versions of this receptor, where Asn 111 in the third transmembrane-spanning domain is replaced with glycine (N111G) or alanine (N111A) (see Fig. 1A), produces constitutive activation of the G␣ q -phospholipase C-␤1-inositol trisphosphate signaling pathway (10). An AT 1A receptor (termed NHA-AT1A), which has an amino-terminal HA epitope tag (YPYDVPDYA), and a truncated mutant (NHA-TK325), where 34 amino acids are removed from the carboxyl terminus, have been described previously (13). Mutated versions of the NHA-AT1A receptor with reduced AngII-stimulated phosphorylation (S335/T336A) (14) and a triple mutant (S331/ S338/S348A) (15) were generated by site-directed mutagenesis and confirmed by sequencing.
Plasmid DNA (0.6 g/well) for the various receptor constructs was mixed with LipofectAMINE (4.8 l/well) and used to transfect CHO-K1 cells in 12-well cell culture plates at ϳ80% confluence, as described previously (13). After 48 h, cells were tested for binding of 125 I-AngII to determine cell surface receptor expression or processed for receptor phosphorylation. The signaling capacity of cells expressing the various receptor constructs was determined by measuring the generation of inositol phosphates in response to the peptide ligands, [Sar 1 ]AngII and [Sar 1 ,Ile 4 ,Ile 8 ]AngII, and the non-peptide antagonist, EXP3174, as described previously (10,12).
Immunoprecipitation and Determination of Receptor Phosphorylation-Methods for phosphorylating and immunoprecipitating transiently transfected, epitope-tagged AT 1A receptors have been described (13). Briefly, transfected cells in 12-well plates were serum-starved for 16 h, loaded with [ 32 P]P i (200 Ci/ml), and stimulated by the agonist AngII or the non-signaling analog, [Sar 1 ,Ile 4 ,Ile 8 ]AngII, for 10 min at 37°C. After stimulation, the plates were placed on ice, washed twice with 1 ml/well of Hanks' buffered salt solution (4°C), and the cells solubilized with 300 l/well of a lysis buffer containing phosphatase inhibitors (13). The cell lysates were centrifuged at 14,000 ϫ g for 15 min and precleared by the addition of bovine serum albumin and protein A-agarose (1 h, 4°C). The epitope-tagged AT 1A receptors were immunoprecipitated from the precleared lysates by adding 2 g of affinity-purified 12CA5 or 1D4 monoclonal antibody and 20 l of protein A-agarose (50% suspension) followed by overnight agitation at 4°C. The immunoprecipitates were washed five times, resuspended in 55 l of a urea-based SDS sample buffer, heated at 60°C for 15 min, and resolved by 10% SDS-polyacrylamide gel electrophoresis. Gels were fixed, dried, and phosphorylated bands detected and quantified by phosphoimaging. Phosphoimaging data were normalized for receptor expression by transfecting parallel plates of CHO-K1 cells with the various constructs and performing AngII radioreceptor-binding assays (16). Binding assays were performed for 5 h at 4°C to prevent receptor internalization.
Measurement of AT 1A Receptor Internalization-CHO-K1 cells, transiently transfected with wild type or constitutively active AT 1A receptors in 12-well plates, were incubated with 125 I-AngII (0.4 nM) for 2, 5, 10, and 20 min at 37°C. Internalization was terminated, and cell surface-bound 125 I-AngII was removed by acid washing, while internalized 125 I-AngII-receptor complexes were harvested with a 0.25 M NaOH, 0.25% SDS solution. Internalization kinetic curves were obtained by expressing the acid-insensitive radioactivity (internalized receptors) as percentage of the total binding (acid-insensitive ϩ acid-sensitive) for each well. The percentage of internalized receptors was plotted against time and analyzed as a one-phase exponential association using Graph-Pad Prism. The t 1/2 (in min) to reach a maximal level of internalization (Y max , in %) was determined for each association curve.
Alternatively, the capacity of AngII and [Sar 1 ,Ile 4 ,Ile 8 ]AngII to promote internalization was examined by adding the unlabeled peptides (100 nM AngII or 30 M [Sar 1 ,Ile 4 ,Ile 8 ]AngII) to transfected cells for 10 min at 37°C. Surface-bound ligands were removed with a gentle acid wash (50 mM sodium citrate, 0.2 mM sodium phosphate, 90 mM NaCl, 0.1% bovine serum albumin, pH 5.0; 10 min, 4°C), which does not effect subsequent receptor binding (17), and then a radioreceptor binding assay was performed (5 h at 4°C) to measure receptors remaining at the cell surface. Internalized receptors were expressed as a percentage loss of cell surface binding compared with cells not exposed to AngII or [Sar 1 ,Ile 4 ,Ile 8 ]AngII.

RESULTS
Receptor Expression and Signaling-Cell surface expression of the receptor constructs used in this study was determined by 125 I-AngII competitive binding assays. As previously observed (10,12,13), wild type and mutant receptors displayed low nanomolar affinity toward AngII; receptor densities (mean Ϯ S.D.; n), calculated by the method of Swillens (18) Fig. 1B is the inositol phosphate production by the various receptors in response to both peptide and non-peptide ligands. As previously observed (10), the N111G-1D4 mutant showed significant constitutive activation, which was prevented by the non-peptide ligand, EXP3174. All constructs were maximally activated by the full agonist, [Sar 1 ]AngII, while only N111A-1D4 (partially) and N111G-1D4 (maximally) responded to [Sar 1 ,Ile 4 ,Ile 8 ]AngII stimulation. The two wild type receptors (WT-1D4 and NHA-AT1) and a receptor truncated at the carboxyl terminus (NHA-TK325) show similar inositol phosphate activation profiles.
Basal and AngII-stimulated Phosphorylation of Constitutive AT 1A Receptors-Receptor phosphorylation was determined in CHO-K1 cells expressing 1D4 epitope-tagged wild type (WT-1D4) or constitutively active mutants (N111A-1D4 and N111G-1D4) of the rat AT 1A receptor. Cells were loaded with [ 32 P]P i and treated with vehicle or the agonist, AngII (100 nM), followed by immunoprecipitation of the receptor using monoclonal antibody specific for the epitope tag. Controls for the phosphorylation assay included a HA-tagged wild type AT 1A receptor (NHA-AT1A, positive control) and a carboxyl terminus truncated version (NHA-TK325, negative control), which we have previously characterized (13). As expected (Fig. 2, top panel), the full-length NHA-AT1A receptor displayed a basal level of phosphorylation that was increased markedly by AngII stimulation, while the truncated NHA-TK325 receptor, which lacks carboxyl-terminal serine and threonine residues, showed a decrease in basal phosphorylation that was not enhanced by AngII treatment. Similar to NHA-AT1A, the wild type 1D4tagged AT 1A receptor (WT-1D4) exhibited a basal level of phosphorylation that could be increased by AngII stimulation; quantification of this phosphorylation, which was normalized for receptor expression, revealed an approximately 3-fold increase in AngII-induced phosphorylation. Surprisingly, the basal phosphorylation of the constitutively active mutant receptors (N111A-1D4 and N111G-1D4) was not increased, and if anything was slightly decreased, compared with WT-1D4. Moreover, the phosphorylation of these mutants was not increased significantly following AngII stimulation. Binding assays (Fig. 2, middle panel) and Western blot analysis (data not shown) revealed approximately equal amounts of receptor expression, indicating that this difference is not due to dramatic variations in cellular receptor content. Effect of EXP3174 on Constitutive AT 1A Receptor Phosphorylation-Some antagonists are able to inhibit the constitutive activity of GPCRs and these ligands are referred to as inverse agonists (4). EXP3174 is a non-peptide antagonist of the AT 1 receptor and pretreatment with EXP3174 of cells expressing the constitutively active AT 1A mutants (N111A and N111G) reduces the basal inositol phosphate generation by 70 -90% (10,11). Thus, EXP3174 is an inverse agonist of the AT 1 receptor (see also Fig. 1B). We next examined whether conversion of the N111A and N111G receptors back to the basal (R) state by treatment with EXP3174 would allow a subsequent AngIIinduced receptor phosphorylation event to occur. As shown in Fig. 3, EXP3174 treatment (100 nM, 16 h) did not reveal a cryptic AngII-induced phosphorylation of N111A-1D4 or N111G-1D4 receptors; these receptors remained poorly phosphorylated both basally and following AngII stimulation.
Phosphorylation of AT 1A Receptors by [Sar 1 ,Ile 4 ,Ile 8 ]AngII-Constitutively active AT 1A receptors may represent an intermediate transition state (RЈ) between the basal (R) and fully active (R*) forms of the receptor (10, 12). In the wild type receptor, transition to the RЈ state appears dependent upon the [Sar 1 ,Ile 4 ,Ile 8 ]AngII) has a reduced affinity (K D ϭ 300 nM) and is unable to activate inositol phosphate signaling through the wild type receptor, even at concentrations 300 times its K D (10). Interestingly, the constitutive mutant (N111G) has an increased affinity for [Sar 1 ,Ile 4 ,Ile 8 ]AngII (K D ϭ 6 nM) compared with wild type receptor, and this analogue promotes maximal signaling of N111G. If the maximally signaling form (R*) of the receptor is the state targeted for phosphorylation (hereafter termed R P ), then [Sar 1 ,Ile 4 ,Ile 8 ]AngII, which drives the constitutive mutant receptor from RЈ into the fully active R* form, should be expected to promote phosphorylation of the N111G receptor. Conversely, the phosphorylation of the wild type receptor should not be stimulated by [Sar 1 ,Ile 4 ,Ile 8 ]AngII. Remarkably, we observed that 30 M [Sar 1 ,Ile 4 ,Ile 8 ]AngII (ϳ500 times the K D for N111G) was incapable of causing phosphorylation of the constitutively active AT 1A receptors (Fig. 4). This result suggests that the RЈ and R* signaling forms can be differentiated from the phosphorylated form of the receptor, R P . Paradoxically, 30 M [Sar 1 ,Ile 4 ,Ile 8 ]AngII (ϳ100 times the K D ) caused a robust phosphorylation of the wild type AT 1A receptor (ϳ85% of that produced by 100 nM AngII, also ϳ100 times the K D ) (see Fig. 4). This observation also illustrates a distinction between the phosphorylated form, R P , and the active signaling form, R*.  Fig. 5, maximal phosphorylation is achieved by 10 nM AngII (ϳ10 times the K D ) with a half-maximal phosphorylation occurring at 1 nM, which is equivalent to the K D . For [Sar 1 ,Ile 4 ,Ile 8 ]AngII, maximal phosphorylation was achieved by 3 M (ϳ10 times the K D ) with a half-maximal phosphorylation occurring at 300 nM, which approximates the K D . Thus, for both AngII and [Sar 1 ,Ile 4 ,Ile 8 ]AngII, the degree of phosphorylation correlates well with the affinity of the ligands for the wild type receptor.

Characterization of AT
To determine whether AngII and [Sar 1 ,Ile 4 ,Ile 8 ]AngII were phosphorylating similar sites on the wild type AT 1A receptor, we next compared the AngII-and [Sar 1 ,Ile 4 ,Ile 8 ]AngII-induced phosphorylation of wild type and mutant receptors. We (13) and others (14) have previously shown that most, if not all, AngII-induced phosphorylation occurs within the serine/threonine-rich AT 1A carboxyl terminus (see also Fig. 2). The region in the middle of the carboxyl terminus between Thr 332 and Ser 338 (13), in particular Ser 335 Thr 336 (14), is an important site for AngII-stimulated phosphorylation. We have also recently shown that triple mutation of three putative protein kinase C phosphorylation sites at Ser 331 , Ser 338 and Ser 348 to alanine in the carboxyl terminus causes significant decreases in both An-gII-and phorbol ester-induced phosphorylation (15). Hence, we compared the phosphorylation produced by AngII and [Sar 1 ,Ile 4 ,Ile 8 ]AngII on wild type (NHA-AT1A) and mutated (NHA-S335/T336A and NHA-S331/S338/S348A) receptors. As shown in Fig. 6, both AngII and [Sar 1 ,Ile 4 ,Ile 8 ]AngII stimulated a phosphorylation of the wild type receptor (NHA-AT1A); phosphorylation by [Sar 1 ,Ile 4 ,Ile 8 ]AngII was slightly less than that produced by AngII. For AngII stimulation, phosphorylation of the NHA-S335/T336A mutant was reduced to ϳ80% of the wild type receptor, while the NHA-S331/S338/S348A mutant was reduced to ϳ60%. Following [Sar 1 ,Ile 4 ,Ile 8 ]AngII stimulation, the double (NHA-S335/T336A) and triple (NHA-S331/S338/S348A) mutants were phosphorylated to a level about 60% and 50%, respectively, of that observed with the wild type receptor (NHA-AT1A). Therefore, qualitatively, both AngII and [Sar 1 ,Ile 4 ,Ile 8 ]AngII appear to be phosphorylating similar sites within the AT 1A receptor carboxyl terminus.

Receptor Internalization in Response to AngII and [Sar 1 ,Ile 4 ,Ile 8 ]AngII Stimulation-
The capacity of wild type and constitutively active AT 1A receptors to internalize in response to AngII was determined by following the time course of 125 I-AngII endocytosis. Internalization kinetic curves comparing WT-1D4, N111A-1D4 and N111G-1D4 receptor expressing cells are shown in Fig. 7A. As previously observed (19), internalization of 125 I-AngII of the wild type receptor was rapid (t 1/2 , 3.5 min) and robust (Y max , 74.2%). Compared with wild type, the N111A mutant (t 1/2 , 3.5 min; Y max , 66.9%) showed no significant reduction in receptor internalization, while the N111G receptor showed a slightly reduced maximal internalization (Y max , 52.7%) and slower rate (t 1/2 , 6.0 min).
Because [Sar 1 ,Ile 4 ,Ile 8 ]AngII does not contain a tyrosine side chain for labeling with radioactive iodine, a different protocol was used to examine the capacity of this analog to induce receptor internalization. Cells expressing wild type receptors were stimulated with unlabeled AngII (100 nM) or [Sar 1 ,Ile 4 ,Ile 8 ]AngII (30 M) for 10 min at 37°C and then chilled on ice to terminate receptor trafficking. After extensive washing, a gentle acid wash step (17) was used to strip bound ligand from receptors remaining at the cell surface. Equilib-rium 125 I-AngII binding (5 h at 4°C) was then performed to quantify cell surface receptor density and, by comparison to unstimulated controls, the degree of receptor internalization. As shown in Fig. 7B, stimulation by AngII caused 44.7 Ϯ 10.6% (mean Ϯ S.D., n ϭ 4) of AT 1A receptors to be sequestered away from the cell surface. In contrast, [Sar 1 ,Ile 4 ,Ile 8 ]AngII treatment induced very little internalization of the wild type receptor (5.5 Ϯ 3.0%, mean Ϯ S.D., n ϭ 4) or the constitutive receptors (data not shown). DISCUSSION In this study, the phosphorylation and internalization of wild type and mutant AT 1A receptors following stimulation by AngII and an analog, [Sar 1 ,Ile 4 ,Ile 8 ]AngII, was examined. We observed that the constitutively active AT 1A receptors (N111A and N111G) are poor substrates for phosphorylation and that this cannot be modulated by AngII or [Sar 1 ,Ile 4 ,Ile 8 ]AngII stimulation or by prior conversion of the constitutive receptors to the basal state by pretreatment with the inverse agonist, EXP3174. Unexpectedly, the wild type receptor was efficiently phosphorylated by the supposedly inactive analog, [Sar 1 ,Ile 4 ,Ile 8 ]AngII, indicating that the molecular switches required for phosphorylation are distinct from those necessary for G protein-mediated signaling. The fact that AngII, but not [Sar 1 ,Ile 4 ,Ile 8 ]AngII, stimulated the internalization of both wild type and constitutively active AT 1A receptors suggests that the processes of internalization and phosphorylation can also be clearly differentiated. Thus, the AT 1A receptor appears to exist in multiple conformational states, each relating to a distinct transitional stage of receptor activation (i.e. basal, intermediate, signaling, phosphorylation, and internalization). In Fig. 8, we provide a model, which summarizes the proposed Based on the frequently applied two-state model for receptor activation (3), constitutively active GPCRs are thought to attain a conformation that mimics the active (R*) state and should therefore be recognized and phosphorylated by GRKs. Indeed, a number of constitutively active GPCRs, such as the ␣ 2A -adrenergic receptor (20), the ␤ 2 -adrenergic receptor (21), the 5-hydroxytryptamine (5-HT 2C ) receptor (22), rhodopsin (23) and the bradykinin B2 receptor (24), have been reported to display enhanced agonist-independent phosphorylation. In support of the idea that the receptor state that propagates signals to G protein is also the one targeted for phosphorylation, January et al. (25) demonstrated that, for a series of ␤ 2 -adrenergic receptor agonists of varying coupling efficiencies, the degree of agonist strength on the wild type ␤ 2 -adrenergic receptor correlated well with the capacity to mediate phosphorylation, internalization, and desensitization. In contrast to these studies, we observed that the constitutively active AT 1A receptors displayed a basal level of phosphorylation, which was similar to the unstimulated wild type receptor, and that this basal phosphorylation was not increased by stimulation with AngII or the substituted analog, [Sar 1 ,Ile 4 ,Ile 8 ]AngII. The fact that AngII and [Sar 1 ,Ile 4 ,Ile 8 ]AngII can cause maximal inositol phosphate signaling through the constitutively active receptors (10) argues against the assumption that the active, signaling state of GPCRs is the form recognized by GRKs and targeted for phosphorylation.
A distinction between signaling and phosphorylation is also sustained by our discovery that the AngII analog, [Sar 1 ,Ile 4 ,Ile 8 ]AngII, causes robust phosphorylation of the wild type AT 1A receptor. Remarkably, this occurs despite an inability of this analog to promote G␣ q -mediated inositol phosphate signaling through the AT 1A receptor (10). Maximal [Sar 1 ,Ile 4 ,Ile 8 ]AngII-mediated phosphorylation was about 85% of that produced by AngII, and relative to its affinity for the wild type receptor, was as efficacious as AngII at inducing phosphorylation. Moreover, [Sar 1 ,Ile 4 ,Ile 8 ]AngII seemingly mediates phosphorylation at the same sites as AngII in the receptor carboxyl terminus, based on a similar degree of inhibition of phosphorylation for wild type and carboxyl-terminally mutated receptors. Thus, to our knowledge, this represents the first example of an apparently "silent" analog, with respect to signaling, being able to generate almost complete "agonist-like" phosphorylation. Given the robust nature of [Sar 1 ,Ile 4 ,Ile 8 ]AngII-induced phosphorylation as well as the apparent similarity of sites phosphorylated in the AT 1A carboxyl terminus by AngII and [Sar 1 ,Ile 4 ,Ile 8 ]AngII, this strongly suggests that this analog is stabilizing a conformation in the AT 1A receptor that is an excellent substrate for GRK-mediated phosphorylation.
Our observation that the optimal conformation for GRKmediated phosphorylation is different from that which couples the receptor to G protein signaling, corroborates, and can be most directly compared, to a very recent study (26)  tutively active ␣ 1B -adrenergic receptors. In their study, Mhaouty-Kodja et al. (26) investigated the phosphorylation and internalization of ␣ 1B -adrenergic receptors made constitutively active through mutation at two separate sites in the receptor molecule: Asp 142 in the DRY motif at the end of the third transmembrane-spanning domain and Ala 293 in the carboxylterminal region of the third intracellular loop. Mutations at Ala 293 (A293I and A293E) caused constitutive signaling (inositol phosphate production), which correlated well with an enhanced basal (agonist-independent) receptor phosphorylation. In direct contrast, mutations at Asp 142 (D142A and D142T), despite yielding greater levels of constitutive activation compared with the Ala 293 mutants, showed no increase in basal phosphorylation as well as a lack of epinephrine-induced phosphorylation. This result, which is very similar to our data on the phosphorylation of the AT 1A Asn 111 mutants, suggests that multiple conformations exist for receptor activation, only some of which serve as targets for phosphorylation. That both Asp 142 in the ␣ 1B -adrenergic receptor and Asn 111 of the AT 1A receptor are located in the third transmembrane-spanning region, albeit at different levels of the helix, may be relevant to these similar observations and points to a shared mechanism of constitutive activity.
Phosphorylation of the AT 1A receptor carboxyl terminus has been implicated in the mechanism of receptor internalization. Progressive truncation of the AT 1A carboxyl terminus (13,14), or specific mutation of serine and threonine residues in the central region of the receptor tail (Thr 332 -Ser 338 ) to alanine (13), produces a concomitant decrease in both receptor phosphorylation and internalization. It was therefore somewhat surprising in the present study to observe that, despite the lack of AngII-mediated phosphorylation of N111A and N111G, the internalization kinetics for 125 I-AngII of N111A was unaffected and only slightly reduced in the case of N111G. This would suggest that AngII-induced phosphorylation of the AT 1A receptor is not mandatory for internalization. Moreover, given the strong phosphorylation of the wild type receptor by [Sar 1 ,Ile 4 ,Ile 8 ]AngII, in the absence of appreciable internalization, it would appear that phosphorylation is incapable of driving the internalization process. Taken together, these observations support the concept of separate receptor states for receptor phosphorylation and for targeting receptors for endocytosis.
Similarly, clear distinctions can also be drawn between the signaling and internalizing forms of the AT 1A receptor. Specifically, [Sar 1 ,Ile 4 ,Ile 8 ]AngII can produce maximal inositol phosphate signaling through the constitutively active AT 1A receptors (10), suggesting it can promote a conformation in the receptor that approximates the active R*. However, we observed no internalization of constitutive or wild type receptors in response to saturating concentrations (30 M) of this analog. Additionally, we (27) and others (28) have previously reported that another AngII analog, [Sar 1 ,Ile 8 ]AngII, which signals very poorly through the wild type receptor (10), is capable of causing a degree of AT 1A receptor internalization that approximates that of AngII. This separation of signaling and internalization is corroborated by earlier reports, which showed that some G protein uncoupled AT 1A receptors mutants (e.g. D74E, Y302A), with severely compromised signaling to AngII (29,30), displayed almost wild type levels of receptor internalization (19,28,30). Furthermore, we have demonstrated that truncation of the AT 1A carboxyl terminus produces a receptor mutant that couples well to G protein and signals in response to AngII stimulation, but exhibits vastly reduced internalization (16). The literature contains many such examples of GPCR mutants that are uncoupled from signaling but retain a capacity for robust internalization or fully signaling receptors that are compromised with respect to endocytosis. Hence, it appears likely that GPCRs in general are able to attain a conformation(s) that allows specific interaction with components of the internalization machinery while preventing productive coupling to heterotrimeric G proteins.
Important clues as to the role of individual amino acid residues in the octapeptide, AngII, in promoting various receptor states can be gleaned from a comparison of the receptor functions evoked by the AngII analogs, [Sar 1 ,Ile 8 ]AngII and [Sar 1 ,Ile 4 ,Ile 8 ]AngII. For example, both analogs poorly activate inositol phosphate production (10), indicating the crucial role for Tyr 4 and Phe 8 of AngII in specifying agonism, in particular Phe 8 (for full discussion, see Ref. 31). Moreover, both analogs cause robust phosphorylation of the wild type receptor (Ref. 13 and this study), indicating that docking of Tyr 4 and Phe 8 of AngII onto their respective partners in the AT 1A receptor (i.e. Asn 111 in transmembrane helix 3 and His 256 in transmembrane domain 6), is dispensable for promoting a conformation required for phosphorylation. These analogs do differ in that [Sar 1 ,Ile 8 ]AngII (27,28), but not [Sar 1 ,Ile 4 ,Ile 8 ]AngII (this study), causes robust internalization, identifying Tyr 4 in AngII as a crucial determinant of AT 1A receptor endocytosis. Despite the unequivocal importance of the aromatic-amide bond between Tyr 4 in AngII and Asn 111 in AT 1A for receptor activation (31), it is interesting that the constitutively active Asn 111 mutants (N111A and N111G) retain a capacity for AngII-induced internalization. Perhaps other points of contact made between Tyr 4 and the receptor (e.g. Phe 77 , Val 108 , Leu 112 , and Tyr 292 ), suggested by our molecular modeling studies, may be more important for dictating internalization. In regard to inducing phosphorylation, positions other than Tyr 4 and Phe 8 of AngII must be involved, and it will therefore be important to examine the capacity for phosphorylation of AngII analogs substituted at other positions.
In conclusion, data from the present study, as well as accumulating evidence from other GPCRs (32)(33)(34)(35)(36)(37), strongly support the notion of multiple receptor conformational states and the transition of receptors through these states in the process of receptor activation and deactivation. Using constitutively active AT 1A receptor mutants and novel analogs of AngII, which select different transitional phases of receptor activation, has provided a unique insight into the physical separation of these putative conformational states. Activation selective analogs, such as [Sar 1 ,Ile 4 ,Ile 8 ]AngII, represent powerful tools to further dissect the molecular mechanisms that underlie the generation of GPCR signals and their subsequent termination by phosphorylation, internalization, and desensitization.