c-Src Regulates Clathrin Adapter Protein 2 Interaction with ß-Arrestin and the Angiotensin II Type 1 Receptor during Clathrin- Mediated Internalization

Delphine Fessart, May Simaan and Stéphane A. Laporte

Hormones and Cancer Research Unit, Department of Medicine, McGill University, Montréal, Québec, Canada H3A 1A1

Address all correspondence and requests for reprints to: Dr. Stéphane Laporte, Department of Medicine, McGill University, Royal Victoria Hospital, 687 Pine Avenue West, Montréal, Québec, Canada H3A 1A1. E-mail: stephane.laporte{at}mcgill.ca.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ß-Arrestins are multifunctional adapters involved in the internalization and signaling of G protein-coupled receptors (GPCRs). They target receptors to clathrin-coated pits (CCPs) through binding with clathrin and clathrin adapter 2 (AP-2) complex. They also act as transducers of signaling by recruiting c-Src kinase to certain GPCRs. Here we sought to determine whether c-Src regulates the recruitment of AP-2 to ß-arrestin and the angiotensin II (Ang II) type 1 receptor (AT1R) during internalization. We show that the agonist stimulation of native AT1R in vascular smooth muscle cells (VSMCs) induces the formation of an endogenous complex containing c-Src, ß-arrestins and AP-2. In vitro studies using coimmunoprecipitation experiments and a yeast three-hybrid assay reveal that c-Src stabilizes the agonist-independent association between ß-arrestin2 and the ß-subunit of AP-2 independently of the kinase activity of c-Src. However, although c-Src expression promoted the rapid dissociation of AP-2 from both ß-arrestin and AT1R after receptor stimulation, a kinase-inactive mutant of c-Src failed to induce the dissociation of AP-2 from the agonist-occupied receptor. Thus, the consequence of c-Src in regulating the dissociation of AP-2 from the receptor was also examined on the internalization of AT1R by depleting c-Src in human embryonic kidney (HEK) 293 cells using a small interfering RNA strategy. Experiments in c-Src depleted cells reveal that AT1R remained mostly colocalized with AP-2 at the plasma membrane after Ang II stimulation, consistent with the observed delay in receptor internalization. Moreover, coimmunoprecipitation experiments in c-Src depleted HEK 293 cells and VSMCs showed an increased association of AP-2 to the agonist-occupied AT1R and ß-arrestin, respectively. Together, our results support a role for c-Src in regulating the dissociation of AP-2 from agonist-occupied AT1R and ß-arrestin during the clathrin-mediated internalization of receptors and suggest a novel function for c-Src kinase in the internalization of AT1R.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
G PROTEIN-COUPLED RECEPTORS (GPCRs) are integral membrane proteins that are responsible for controlling an array of physiological responses such as phototransduction, olfaction, neurotransmission, vascular tone, cardiac output, and pain. The intensity and the duration of a response are dependent on the balance that exists between mechanisms that regulate the coupling of the receptors to their downstream effectors and those that terminate the signaling. Mechanisms that rapidly turn off signaling include desensitization, whereby receptors become refractory to subsequent stimuli, and internalization, where receptors are removed from the plasma membrane (a process also referred to as sequestration or endocytosis) (1, 2). Internalization of GPCRs can lead to the reestablishment of the cellular response by the recycling of receptors to the plasma membrane, or the prevention of further signaling by targeting receptors for degradation.

Homologous desensitization and internalization of receptors involve common and distinct steps. Agonist stimulation of GPCRs promotes the phosphorylation of receptors by GPCR kinases (GRKs), and the recruitment of nonvisual arrestin proteins (ß-arrestin1 and ß-arrestin2) to the phosphorylated receptors (1). ß-Arrestins bind to the agonist-occupied GPCR to prevent second-messenger signaling and act as adapters to target desensitized receptors to clathrin-coated vesicles (CCVs) at the plasma membrane (i.e. clathrin-coated pits; CCPs) for internalization (3, 4). The involvement of ß-arrestins in the internalization of the ß2-adrenergic receptor (ß2AR) and the angiotensin II (Ang II) type 1 receptor (AT1R) has been demonstrated using different approaches, and more recently by gene disruption and posttranscriptional silencing of ß-arrestins (5, 6). In the case of the ß2AR, ß-arrestins are believed to link the agonist-occupied receptors to CCPs by interacting through their C-terminal domains with both clathrin and the ß-subunit of the heterotetrameric adapter AP-2 complex (ß2-adaptin) (7, 8, 9, 10). Many studies indicate that AT1R can internalize via CCVs in both heterologous and endogenous systems (11, 12, 13, 14, 15), but the underlying mechanisms regulating ß-arrestin-mediated endocytosis of AT1R and other GPCRs still remain poorly understood.

In addition to their roles in GPCR desensitization and internalization, recent evidence indicates that ß-arrestins can also participate in receptor signaling by functioning as adapters for the recruitment of signaling molecules to agonist-occupied receptors (16). For instance, ß-arrestins have been shown to recruit the nonreceptor tyrosine kinase c-Src to the ß2AR (17) and the neurokinin-1 receptor (18). The ß-arrestin-mediated recruitment of c-Src to the agonist-occupied ß2AR regulates the function of the endocytic protein dynamin, a GTPase involved in the fission of CCPs from the plasma membrane (8, 17). Given the dual adapter role for ß-arrestins in the internalization and signaling of GPCRs, we hypothesize that c-Src could also regulate the recruitment of other endocytic proteins like AP-2 to the agonist-induced AT1R/ß-arrestin complex, and consequently impact the early steps of receptor internalization.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ang II Promotes the Formation of an Endogenous Complex Containing c-Src, ß-Arrestin, and AP-2
We have previously shown that the internalization of certain GPCRs, via the clathrin pathway, requires the recruitment of ß-arrestin to the receptor, and the interaction of ß-arrestin with the heterotetrameric AP-2 complex (via its ß-subunit, i.e. ß2-adaptin) (9). More recently, it has been suggested by different groups that ß-arrestins act as signaling adapters for the recruitment of c-Src family kinases to some GPCRs (17, 18, 19). To determine whether c-Src could participate in the association between ß-arrestin and AP-2, we first examined in an endogenous system, whether the agonist stimulation of AT1R promoted the formation of a complex containing c-Src, ß-arrestin, and AP-2. Vascular smooth muscle cells (VSMCs) expressing endogenous AT1R (around 100 fmol/mg of total protein as estimated by radioligand binding experiments; data not shown) were serum starved for 12 h and then either left untreated or treated with Ang II for 5 min at 37 C. Cells were then solubilized and endogenous c-Src was immunoprecipitated. Interacting proteins in the complex were identified by Western blot analysis using antibodies against the {alpha}-subunit of AP-2 and ß-arrestin (Fig. 1Go). Results show that, in the absence of agonist stimulation, little ß-arrestin and no AP-2 complex were detected in the c-Src immunoprecipitates. However, after agonist stimulation of native AT1R, a robust association of both ß-arrestin and AP-2 was observed with c-Src. These results show that the agonist stimulation of AT1R in a native system like VSMCs promotes the formation of an endogenous complex including c-Src, ß-arrestin, and AP-2.



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Fig. 1. Ang II Promotes the Formation of an Endogenous Complex Containing c-Src, ß-Arrestin, and AP-2

VSMCs were left untreated (–) or treated (+) with Ang II (100 nM) for 5 min at 37 C after serum starvation overnight. Endogenous c-Src was immunoprecipitated (IP) with the GD11 antibody, and proteins in the complex were detected by Western blot using c-Src (SRC2, Santa Cruz Biotechnology), ß-arrestins (A1CT), and the {alpha}-subunit of AP-2 (BD Transduction Laboratories) antibodies as described in Materials and Methods. Also presented are blots from whole cell extracts (Total, right panel) probed with the same antibodies. Blots are representative of three independent experiments.

 
c-Src Increases the Association between ß-Arrestin and ß2-Adaptin Independently of Its Kinase Activity
The Ang II-mediated formation of a complex containing ß-arrestin, AP-2, and c-Src in VSMCs, and the recent demonstration that c-Src and ß-arrestin associate with each other when overexpressed in COS-7 cells (20), suggest that c-Src is involved in stabilizing the association between ß-arrestin and AP-2. We therefore reasoned that, by increasing the expression of c-Src in cells, we could induce the agonist-independent association between ß-arrestin and AP-2. To test this possibility, COS-7 cells were transfected with Flag-tagged ß-arrestin2 and increasing amounts of hemagglutinin (HA)-tagged c-Src, and the level of endogenous AP-2 (as assessed by the presence of ß-subunit of the AP-2 complex, i.e. ß2-adaptin) in the ß-arrestin immunoprecipitates was identified by Western blot analysis (Fig. 2AGo). In the absence of c-Src overexpression (Fig. 2AGo, lane 1), no ß2-adaptin was detected in the ß-arrestin immunocomplex. However, increasing the expression of c-Src resulted in the increased formation of a ß-arrestin/AP-2 complex. We next examined whether this c-Src-mediated GPCR-independent association between ß-arrestin2 and AP-2 was dependent on the kinase activity of c-Src. To this end, we overexpressed ß-arrestin2-Flag and ß2-adaptin in COS-7 cells with either wild-type c-Src or a mutant that mimics the activated form of c-Src (c-Src-Y530F) or the kinase dead c-Src-K298R (Fig. 2Go, B and C), and assessed the ability of the different c-Src constructs to promote the association between ß2-adaptin and ß-arrestin. We first determined the relative level of kinase activity of the different c-Src constructs by probing the total lysates from cells expressing wild-type and c-Src mutants using an antiphosphotyrosine antibody (Fig. 2BGo). The expression of wild-type c-Src and c-Src-Y530F had similar effects on the pattern of protein phosphorylation, whereas almost no phosphorylation of proteins was detected in cells overexpressing c-Src-K298R, suggesting that at the levels of c-Src and c-Src-Y530F expression, both kinases had a similar activity in cells. Subsequently, these different constructs were overexpressed in COS-7 cells, and the amounts of AP-2 and c-Src associated to the immunopurified ß-arrestin2 complexes were analyzed. Although we found that all c-Src constructs were able to associate with ß-arrestin2, only wild type c-Src and the K298R mutant promoted the formation of a ternary complex that included ß2-adaptin and ß-arrestin (Fig. 2CGo). These results imply that the kinase activity of c-Src is not required to promote the agonist-independent association of AP-2 with ß-arrestin2.



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Fig. 2. c-Src Overexpression Increases the Association between ß-Arrestin and AP-2 Independently of the c-Src Kinase Activity

A, COS-7 cells were transiently transfected with Flag-ß-arrestin2 and increasing amounts of HA-c-Src. ß-Arrestin was immunoprecipitated (IP) with Flag antibody (Sigma) and the amount of endogenous AP-2, as assessed by the presence of ß2-adaptin in the ß-arrestin immunoprecipitates was analyzed by Western blot using a ß2-adaptin (BD Transduction Laboratories) antibody. Whole cell extracts (Total) were also blotted for detecting the level of c-Src expression using the HA (Santa Cruz Biotechnology) and ß2-adaptin (BD Transduction Laboratories) antibodies. B, COS-7 cells were transfected with Flag-ß-arrestin2, ß2-adaptin and pcDNA3.1 (Mock) and either HA-c-Src, HA-c-Src-Y530F or HA-c-Src-K298R. Whole cell extracts (Total) were analyzed by Western blot (WB) with the antiphosphotyrosine (P-Tyr) antibody 4G10 (Upstate Biotechnology). C, Cells were transfected as in (B), and Flag-ß-arrestin was IP with the Flag antibody (Sigma), and complexes were analyzed by Western blot for the detection of ß-arrestin, ß2-adaptin, and c-Src expression using the antibodies against Flag (Sigma), ß2-adaptin (BD Transduction Laboratories), and HA (Santa Cruz Biotechnology), respectively. Whole cell extracts (Total) were also blotted with anti-HA (Santa Cruz Biotechnology) and anti-ß2-adaptin (BD Transduction Laboratories) antibodies, for detection of the different c-Src constructs and the ß2-adaptin, respectively. Data are representative of at least three independent experiments.

 
To verify that c-Src directly contributed to the formation of a ternary complex with ß-arrestin2 and ß2-adaptin, and also to confirm that this complex formation is independent of c-Src kinase activity, we used a modification of the yeast two-hybrid system (yeast three-hybrid). We have previously shown using a similar assay that the interaction between ß-arrestin2 and ß2-adaptin could be detected (8, 9). We expressed ß-arrestin2 fused to the DNA binding domain (DBD) of GAL4 and ß2-adaptin fused to the activation domain (AD) of GAL4, and assessed the effect of c-Src expression on the transactivation of the auxotrophic HIS3 and the reporter lacZ genes (Fig. 3CGo). The expression vectors for ß-arrestin2 and the ß2-adaptin were cotransformed into yeast strain PJ69-4a with the empty vector p426-ADH or with the p426-ADH-c-Src mutant (c-Src-G2A, -Y416F, -Y527F). This mutant lacks the myristoylation site, to reduce its membrane association, and is mutated on tyrosines 416 and 527 to prevent tyrosine phosphorylation, and hence the activation of c-Src (21). Yeasts were transformed with the expression vector for ß-arrestin2 alone (Fig. 3AGo, lane A), or with ß2-adaptin (lane B), c-Src (lane C) or ß2-adaptin and c-Src together (lane D). Cells were serially diluted and spotted onto plates lacking leucine, tryptophan, and uracil supplemented or not with histidine (Fig. 3AGo, left and right panels). As shown here, the overexpression of ß-arrestin2 and ß2-adaptin is sufficient to induce yeast growth on selective media lacking histidine (Fig. 3AGo, right panel, lane B). The expression of c-Src with both ß-arrestin2 and ß2-adaptin promoted the faster growth of yeasts (Fig. 3AGo, right panel, compare lanes D and B), suggesting that c-Src stabilizes the ß-arrestin/ß2-adaptin interaction. We also quantified the interaction between ß-arrestin2 and ß2-adaptin using a lacZ reporter assay. Our results show that expression of c-Src with ß-arrestin2 and ß2-adaptin induced a 2- and 4-fold increase in the ß-galactosidase activity as compared with yeasts expressing ß-arrestin and ß2-adaptin, or yeasts expressing ß-arrestin and c-Src, respectively (Fig. 3BGo, D vs. B and D vs. C, respectively). These results indicate that the expression of c-Src in yeast stabilizes the ternary complex containing ß-arrestin2 and ß2-adaptin. As expected, we did not find that the expression of the c-Src mutant increased the tyrosine phosphorylation of total yeast protein (data not shown). Altogether, these results demonstrate that the interaction between ß-arrestin and ß2-adaptin can be stabilized in the presence of c-Src, and support our previous observations that the kinase activity of c-Src is not necessary to induce the agonist-independent formation of the complex.



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Fig. 3. c-Src Increases the Interaction between ß-Arrestin and ß2-Adaptin in Yeast

A, DNA encoding the hybrid protein ß-arrestin2 (GAL4 DBD-ßarr2) was transformed in the yeast strain PJ69-4a alone (lane A), or with the hybrid protein ß2-adaptin (GAL4 AD ß2-Ad) (lane B), c-Src (lane C), or ß2-adaptin and c-Src together (lane D) as described in Materials and Methods. Transformants were serially diluted (at an A600 of 0.1, 0.002, 0.004, respectively) and 10 µl of cell suspension were spotted onto selective medium lacking leucine, tryptophan and uracil (–Leu/–Trp/–Ura), or lacking leucine, tryptophan, uracil and histidine (–Leu/–Trp/–Ura/–His). Protein-protein interaction was assessed for histidine complementation in presence of 2.5 mM 3-AT (A) and for ß-galactosidase activity (B). ß-Galactosidase activity is expressed as Miller units as described in Materials and Methods, and represents the mean ± SEM of three independent transformations in triplicates. C, Schematic representation of ternary complex formation between the two hybrid proteins, ß-arrestin2 and ß2-adaptin, and the bridge protein c-Src. Results show that the expression of c-Src with the two-hybrid proteins (column D) increases the ß-galactosidase activity by 4- and 2-fold as compared with conditions when ß-arrestin2 and c-Src (column C), or ß-arrestin2 and ß2-adaptin (column B) were expressed together. ß2-Adaptin expression alone or with c-Src did not increase significantly the ß-galactosidase activity as compared with conditions expressing ß-arrestin2 and c-Src (data not shown). **, P < 0.01; ***, P < 0.001 determined by one-way ANOVA, and was considered highly significant.

 
The c-Src-Mediated Formation of a ß-Arrestin/AP-2 Complex Requires an Intact ß2-Adaptin Binding Domain on ß-Arrestin
We have previously shown that ß2-adaptin binds directly ß-arrestin2 through a critical arginine residue (R396) present in the C-terminal domain of ß-arrestin (8). This region also contains a motif for the binding of clathrin (LIEF, residues 374–377) (7, 8, 9, 22). Therefore, we used different ß-arrestin2 mutants impaired in ß2-adaptin binding (ßarr2 R396A), impaired in both clathrin and ß2-adaptin binding (where the hydrophobic residues of LIEF motif were substituted for alanines, ßarr2-R396A,-AAEA) or a truncated form of ß-arrestin2 lacking both sites (ßarr2-T372), and assessed the ability of c-Src to promote the agonist-independent binding of ß2-adaptin to the different ß-arrestin mutants. COS-7 cells were transfected with either Flag-tagged ß-arrestin2 or mutants, with or without HA-c-Src, and the amount of endogenous AP-2 in the ß-arrestin immunoprecipitates was determined by Western blot analysis (Fig. 4Go). As previously observed and shown here, the overexpression of HA-c-Src induced the association of AP-2 with ß-arrestin2 (Figs. 2Go and 4Go). However, c-Src failed to promote the robust association of AP-2 to the different ß-arrestin2 mutants as compared with wild-type ß-arrestin2 (Fig. 4Go). These results demonstrate that the c-Src-induced formation of a ß-arrestin/AP-2 complex requires an intact ß2-adaptin binding site on ß-arrestin2 C-terminal domain.



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Fig. 4. Effect of c-Src expression on AP-2 Association with ß-Arrestin Mutants Lacking ß2-Adaptin and/or Clathrin Binding Sites

A and B, COS-7 cells were transiently transfected with either Flag-ß-arrestin2 or Flag-ß-arrestin2-R396A, Flag-ß-arrestin2-R396A/AAEA, Flag-ß-arrestin2-T372, with or without HA-c-Src. ß-Arrestin2 wild type or mutants were immunoprecipitated with an anti-Flag antibody (Sigma), and the endogenous AP-2 in the immunoprecipitates was detected by Western blot using ß2-adaptin (BD Transduction Laboratories) antibody. Whole cell extracts (Total) were blotted with anti-HA (Santa Cruz Biotechnology) antibody for c-Src detection, and with the same anti-ß2-adaptin antibody as described above. B, Results are expressed as the fold increase association of AP-2 with ß-arrestin2 in presence of c-Src (+) as compared with conditions where c-Src was not overexpressed (–), and represent the mean ± SEM of at least three independent experiments. *, P < 0.05; **, P < 0.01 as compared with wild-type ß-arrestin determined by one-way ANOVA test.

 
The Kinase Activity of c-Src Is Required to Promote the Dissociation of AP-2 from ß-Arrestin2 and Agonist-Occupied AT1R
We have previously shown for some GPCRs internalizing via CCVs, such as the ß2AR that the recruitment of AP-2 to ß-arrestin and to the receptor required agonist activation (9). We therefore first verified whether the agonist stimulation of AT1R could induce the recruitment of AP-2 to ß-arrestin and the receptor (Fig. 5Go). COS-7 or human embryonic kidney (HEK) 293 cells were used to express Flag-tagged ß-arrestin2 and wild-type AT1R, or Flag-tagged AT1R alone. Cells were serum starved and then stimulated with Ang II for different periods of time (Fig. 5Go, A and B, respectively). ß-Arrestin2 in COS-7 cells or AT1R in HEK 293 cells was then immunoprecipitated and the amount of endogenous AP-2 in the complexes, as detected by the presence of ß2-adaptin, was determined by Western blot analysis (Fig. 5Go, A and B, lane 1–3). In unstimulated cells, little association of AP-2 to ß-arrestin and AT1R was observed, consistent with what we observed for other GPCRs (9). Stimulation of receptors induced the recruitment of AP-2 to both ß-arrestin and the receptor in a time-dependent manner reaching a maximum after 5 min of agonist treatment (Fig. 5Go, A and B, lanes 1–3). Association of AP-2 with ß-arrestin was transient and the complex was lost after 15 min of agonist treatment (data not shown). The time frame of AP-2 recruitment to AT1R in HEK 293 cells (Fig. 5BGo) was similar to that observed for the interaction between ß-arrestin and AP-2 in COS-7 cells (Fig. 5AGo) or in HEK 293 cells (data not shown). Together, these results suggest that AP-2 is recruited to the same AT1R/ß-arrestin complex. Indeed, in a different set of experiments, when the agonist-occupied AT1R was immunoprecipitated, both ß-arrestin and AP-2 were found in the same receptor complex (data not shown).



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Fig. 5. c-Src Promotes the Dissociation of AP-2 from the Agonist-Occupied AT1R and ß-Arrestin

COS-7 cells were transfected with AT1R and Flag-ß-arrestin2 with or without c-Src (A), or HEK 293 cells were transfected with Flag-AT1R with or without c-Src and c-Src-K298R (B and C). Cells were serum-starved 12 h before being stimulated with Ang II (1 µM) for the indicated time, and ß-arrestin2 (A) or AT1R (B and C) were immunoprecipitated (IP) using an anti-Flag antibody (Sigma). Immunoprecipitates were analyzed by Western blot for the detection of associated endogenous AP-2 using an antibody against ß2-adaptin (BD Transduction Laboratories). Whole cell extracts (Total) were also probed for the detection of c-Src using the GD11 antibody (Upstate) and endogenous AP-2 using the BD Transduction Laboratories antibody. Data represent the relative amount of ß2-adaptin found in AT1R and ß-arrestin2 immunoprecipitates after agonist-stimulation of receptors after normalizing for equal amounts of ß2-adaptin present in total proteins, and are the mean ± SEM of three independent experiments.

 
To elucidate the mechanism by which c-Src regulates the agonist-mediated formation of this endocytic complex, we next assessed the effect of overexpressing c-Src on the association of AP-2 with ß-arrestin and AT1R after receptor stimulation. When c-Src was overexpressed, AP-2 was already detected in the ß-arrestin immunoprecipitates in the absence of agonist treatment, and almost amounted to levels of stimulated receptors in the condition where c-Src was not overexpressed (Fig. 5AGo, compare lanes 3 and 4). Agonist stimulation did not promote further recruitment of AP-2 to ß-arrestin but instead induced the rapid dissociation of the complex, which was complete after 5 min of agonist treatment. The overexpression of c-Src also promoted a basal association of AP-2 with AT1R. Similar to the experiment with ß-arrestin, Ang II stimulation induced the rapid dissociation of AP-2 from the receptor (Fig. 5BGo). AP-2 interaction with AT1R was completely lost after 5 min of Ang II stimulation. Our data indicate that c-Src overexpression promotes the rapid dissociation of AP-2 from agonist-occupied AT1R and ß-arrestin complexes.

To determine whether the kinase activity of c-Src is involved in regulating the dissociation of AP-2 from the agonist-stimulated receptor, we overexpressed the kinase inactive c-Src-K298R and assessed the recruitment of AP-2 to the agonist-occupied AT1R. HEK 293 cells were transfected with Flag-AT1R and c-Src-K298R, and the amount of endogenous AP-2 in the receptor immunoprecipitates was determined by Western blot analysis (Fig. 5CGo). The overexpression of c-Src-K298R induced a basal association of AP-2 with the receptor, similar to that observed with wild-type c-Src (compare Fig. 5Go, C and B). However, whereas c-Src induced the rapid dissociation of AP-2 from agonist-occupied receptor (Fig. 5BGo), the stimulation of AT1R in cells expressing c-Src-K298R promoted both an increased and sustained association of AP-2 with the receptor. Altogether, these results indicate that the kinase activity of c-Src promotes the dissociation of AP-2 from ß-arrestin and AT1R only when receptors are activated by the ligand.

Depletion of c-Src Affects the Redistribution of Agonist-Bound AT1R Inside the Cell and the Dissociation of AP-2 from the Receptor and ß-Arrestin
We next examined the effect of depleting c-Src on the agonist-mediated endocytosis of AT1R using a small interfering RNA (siRNA) strategy. HEK 293 cells were transfected with HA-AT1R and siRNA for human c-Src (siRNA-c-Src), and the ability of siRNAs to silence the expression of c-Src was compared with cells transfected with the receptor and either pcDNA3.1 (Mock) or siRNA for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (siRNA-GAPDH) (Fig. 6AGo). siRNA-c-Src reduced the expression of endogenous c-Src by more than 65% and 55%, respectively, compared with either mock or the nonsilencing control siRNA-GAPDH. The silencing of c-Src showed significant selectivity and did not affect the expression of ß2-adaptin and AT1R (Fig. 6AGo, and results not shown, respectively). We next investigated using confocal microscopy, the effect of siRNA-c-Src on the early events of AT1R internalization after agonist stimulation. HEK 293 cells were transfected with HA-AT1R and green fluorescent protein (GFP)-ß2-adaptin as a marker of AP-2 complex (22, 23) with or without siRNA-c-Src, and cells were labeled at 14 C for receptor detection. The redistribution of labeled-AT1R and GFP-ß2-adaptin was visualized after a 5 min treatment of cells with Ang II at 37 C. In the absence of agonist, AT1R was found at the plasma membrane in punctuate structures, and little colocalization of the receptor with GFP-ß2-adaptin was observed (Fig. 6BGo, upper left panel). Agonist treatment of cells for 5 min induced the rapid endocytosis of labeled-AT1R from the plasma membrane into intracellular vesicles with no noticeable redistribution of ß2-adaptin fluorescence. In c-Src-depleted cells and in the absence of Ang II stimulation, AT1R was found at the plasma membrane and did not colocalize with GFP-ß2-adaptin even though qualitatively more ß2-adaptin fluorescence was detected at the plasma membrane. After agonist stimulation of receptors for 5 min, AT1R was found mainly at the plasma membrane and mostly colocalized with GFP-ß2-adaptin (Fig. 6BGo, lower right panel). The higher incidence of AT1R colocalization with ß2-adaptin at the plasma membrane in c-Src-depleted cells is consistent with a role of c-Src in regulating the dissociation of AP-2 from the receptor. To verify that c-Src depletion affected the dissociation of AP-2 from AT1R, we performed coimmunoprecipitation experiments on cells that were transfected using the same conditions as for the confocal experiments (Fig. 6CGo). Results show that in cells with reduced c-Src expression, a robust increase in the agonist-mediated association of AP-2 with the immunopurified AT1R was observed, as compared with nondepleted cells (mock) and GAPDH-depleted cells. We also assessed the effect of reducing c-Src expression on AT1R internalization by radio-ligand binding assay using 125I-AngII. As shown in Fig. 6DGo, agonist stimulation of HEK 293 cells expressing the AT1R alone promoted the rapid internalization of receptor-ligand complex inside the cell, which reached a maximum after only 5–10 min of agonist stimulation. The nonsilencing control siRNA-GAPDH did not affect the rate or the extent to which AT1R internalizes. However, reducing the endogenous expression of c-Src delayed receptor endocytosis, and maximal internalization was reached only after 10–15 min of Ang II stimulation (Fig. 6DGo). Internalization was significantly reduced in c-Src-depleted cells as compared with nondepleted cells (AT1R alone) at 2 and 5 min of receptor stimulation (34 ± 3% vs. 52 ± 2%; and 41 ± 2% vs. 63 ± 4%, respectively). The half-time of AT1R internalization (the time to which 50% of receptors were internalized) in c-Src-depleted cells increased 2-fold as compared with nonsilenced cells (Fig. 6DGo). The delay in the initial rate of AT1R internalization observed in c-Src-depleted cells suggests that c-Src is involved in the early events of receptor internalization.



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Fig. 6. Depleting c-Src in Cells Affects the Agonist-Mediated Internalization of AT1R and Its Association with AP-2

A, HEK 293 cells were transiently transfected with HA-AT1R with pcDNA3.1 (Mock), siRNA-GAPDH, or siRNA-c-Src. Seventy-two hours after transfection, total cell lysates were analyzed by Western blot for the expression of endogenous c-Src using the GD11 (Upstate) and the ß2-adaptin (BD Transduction Laboratories) antibodies. B, HEK 293 cells were transfected with HA-AT1R and GFP-ß2-adaptin, and either pcDNA3.1 (upper panels) or siRNA-c-Src (lower panels). Cells were serum starved for 12 h before being incubated with anti-HA antibody (12CA5, Roche) and with a secondary goat antimouse antibody conjugated to AlexaFluor 568 (Molecular Probes) as described in Materials and Methods. Cells were then washed, and were either left untreated or treated with Ang II (1 µM) for 5 min at 37 C. The distribution of HA-AT1R and GFP-ß2-adaptin was visualized by confocal microscopy. For each experiment, 25 different fields containing one to two cells were analyzed. Shown are representative images of receptor immunofluorescence (red) and GFP-ß2-adaptin fluorescence (green) of three independent experiments. Colocalization (yellow) of GFP-ß2-adaptin with the receptor is indicated by arrows. C, HEK 293 cells were transiently transfected with HA-AT1R with either pcDNA3.1 (Mock), siRNA-c-Src, or siRNA-GAPDH. Cells were serum-starved as previously described before being stimulated with Ang II (1 µM) for 5 min. AT1R was immunoprecipitated (IP) using an anti-HA antibody (Roche), and the immunoprecipitates were analyzed by Western blot for the detection of associated AP-2 using the ß2-adaptin antibody (BD Transduction Laboratories). Whole cell extracts (Total) were also probed for the expression of endogenous c-Src using the GD11 (Upstate) and the ß2-adaptin (BD Transduction Laboratories) antibodies. Blots are representative of two independent experiments. D, For receptor internalization, cells were transfected with AT1R alone ({blacksquare}) and with either siRNA-c-Src ({Delta}) or siRNA-GAPDH ({square}). Cells were incubated with [125I]-Ang II (0.11 nM) at 37 C for the different period of time, and the percent of receptor internalization was calculated as described in Materials and Methods. Data are the mean ± SEM of three to five independent experiments and were analyzed using Prism4 (GraphPad 4 Software). *, P < 0.05 siRNA-c-Src vs. AT1R alone determined by an unpaired t test.

 
To substantiate the role of c-Src in regulating the association of AP-2 with AT1R/ß-arrestin complexes, we depleted c-Src expression in an endogenous system like the VSMCs. Cells were first treated with control antisense DNA (CTL-1) or antisense DNA for c-Src (AS-1), and the amount of c-Src expression was assessed. As shown in Fig. 7AGo, AS-1 selectively decreased by more than 60% the expression of c-Src, whereas the CTL-1 had no effect on the expression of the protein. We next performed coimmunoprecipitation experiment to assess the effect of reducing c-Src expression on AP-2 association with endogenous ß-arrestin after AT1R stimulation. VSMCs transfected with either AS-1 or CTL-1 were left unstimulated or stimulated with Ang II for 5 min, and the level of AP-2 (as assessed by the presence of the ß2-adaptin) in the ß-arrestin immunoprecipitates was evaluated by Western blot analysis (Fig. 7BGo). Ang II stimulation induced a rapid association of AP-2 with ß-arrestin. However, the extent to which AP-2 associated with ß-arrestin was greater in c-Src-depleted VSMCs than in control cells. All together, these results support the role of c-Src in regulating the dissociation of AP-2 from AT1R and ß-arrestin during the internalization of receptors.



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Fig. 7. Depleting c-Src in VSMCs Increases the AngII-Mediated AP-2 Association with ß-Arrestin

A, VSMCs cells were transfected or left untransfected (Mock) with either antisense oligonucleotides for the rat c-Src (AS-1) or control oligonucleotides (CTL-1). Thirty-six hours after transfection, total cell lysates from VSMCs were analyzed by Western blot for the expression of endogenous c-Src using the GD11 (Upstate) and ß2-adaptin (BD Transduction Laboratories) antibodies. B, VSMCs were transfected with oligonucleotides for 36 h in serum-free medium as described in Materials and Methods. Cells were then stimulated with Ang II (1 µM) for 5 min, and ß-arrestin was immunoprecipitated (IP) using the A1CT antibody. Proteins in the immunoprecipitated complexes were analyzed by Western blot for the detection of associated AP-2 using the ß2-adaptin antibody. Whole cell extracts (Total) were also probed for the detection of endogenous c-Src using the GD11 (Upstate), ß-arrestin (A1CT) and ß2-adaptin antibodies. Shown in A and B are representative blots of four independent experiments. Data were quantified and presented as the relative amount of c-Src found in cells after normalizing to the total amount of ß2-adaptin (A), and the fold increase over basal of the associated ß2-adaptin with ß-arrestin (B). *, P < 0.05 AS-1 vs. CTL-1 as determined by one-tail paired t test.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Here we show that AP-2 is recruited to ß-arrestin and AT1R after receptor stimulation, extending previous studies that AT1R internalizes through the clathrin pathway (11, 12, 13, 14, 15). More importantly, we provide evidence that c-Src regulates the binding of AP-2 to ß-arrestin and agonist-occupied AT1R. Indeed, we demonstrate that c-Src can form a stable complex with AP-2 and ß-arrestin, and that its kinase activity is required to promote the agonist-mediated dissociation of AP-2 from both ß-arrestin and AT1R during receptor internalization. In support of these results is our finding that depleting c-Src expression in cells increases the binding of AP-2 with the agonist-stimulated AT1R and ß-arrestin and prevents the rapid redistribution of the agonist-bound receptor inside the cell.

The recruitment of AP-2 to ß-arrestin is mediated by the agonist stimulation of GPCRs (9). Interestingly, c-Src overexpression was sufficient to promote the stable association between AP-2 and ß-arrestin, and AT1R without inducing receptor endocytosis (results not shown). This effect was independent of the kinase activity of c-Src but required the presence of an intact ß2-adaptin binding site on ß-arrestin. It was recently suggested, based on the crystal structure of ß-arrestin, that the activation of receptors is necessary to induce favorable conformational changes in ß-arrestin required to unveil the ß2-adaptin binding site in the C-terminal domain of ß-arrestin (24). Our results suggest that c-Src binding to ß-arrestin induces a conformational change in ß-arrestin allowing the agonist-independent binding of AP-2 to ß-arrestin and AT1R. Interestingly, a mutant that mimics the activated form of c-Src (c-Src-Y530F) was unable to promote the association of AP-2 to ß-arrestin. One explanation may be that c-Src-Y530F cannot induce the appropriate conformation in ß-arrestin to promote the binding of ß2-adaptin to ß-arrestin, even though we found that c-Src-Y530F still associated with ß-arrestin. Another possibility is that this c-Src mutant, because of its potentially increased kinase activity, induces the phosphorylation of protein(s) involved in the dissociation of AP-2 from ß-arrestin, thus preventing the formation of a ternary complex. This latter possibility raises the question of what proteins c-Src is targeting? One potential candidate is the ß-subunit of the AP-2 complex itself. It was recently reported that EGFR activation could induce the tyrosine phosphorylation of ß2-adaptin (25). Whether c-Src phosphorylates ß2-adaptin or other endocytic proteins after AT1R stimulation, and to what extent this phosphorylation plays a role in regulating the dissociation of AP-2 from ß-arrestin and the receptor remain to be determined.

The recruitment of signaling effectors into CCPs has been shown to affect components of the clathrin coat and to regulate the efficient agonist-dependent endocytosis of receptors. The presence of c-Src into CCPs affects coat proteins such as dynamin and clathrin (17, 26). For instance, EGFR stimulation induces the c-Src-dependent phosphorylation of dynamin and clathrin, which plays a role in EGFR endocytosis (26, 27). For GPCRs such as the ß2AR, the agonist-dependent binding of ß-arrestin to receptors induces the formation of a complex containing ß-arrestin and c-Src (28). The ß-arrestin-mediated recruitment of c-Src to ß2AR brings the kinase in close proximity of dynamin, and regulates the function of dynamin through phosphorylation (17). Our results suggest another function for the ß-arrestin-mediated recruitment of c-Src. In this paradigm, the agonist stimulation of AT1R promotes the translocation of ß-arrestin to the receptor, and the recruitment of both c-Src and AP-2 to the AT1R (Fig. 8Go). The binding of c-Src to ß-arrestin and AP-2 would then stabilize the endocytic complex and allow the receptor to be efficiently targeted to the CCP. The Ang II-mediated recruitment of c-Src into the CCP would serve to regulate the dissociation of AP-2 from ß-arrestin and may phosphorylate other proteins of the coat to ensure efficient endocytosis of AT1R. In this regard, it will be of interest to examine whether the stimulation of AT1R triggers the phosphorylation of dynamin and clathrin, and to which extent these c-Src-mediated events contribute in regulating the dissociation of AP-2 from the receptor/ß-arrestin complex, and affect the internalization of AT1R and/or other GPCRs that internalize in a ß-arrestin- and a clathrin-dependent manner.



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Fig. 8. Proposed Model for the Role of c-Src in the Regulation of the Interaction between AP-2, AT1R, and ß-Arrestin during Receptor Internalization

Agonist stimulation of AT1R promotes the recruitment of a ternary complex containing c-Src, AP-2 and ß-arrestin. The complex would then be targeted to CCPs, and the presence of multiple receptor complexes in addition to the recruitment of other endocytic proteins into the CCP would trigger the internalization of the coated vesicle. During AT1R internalization, c-Src could promote the dissociation of AP-2 from the receptor/ß-arrestin complex to allow the recycling of AP-2 for further rounds of coat formation and AT1R targeting to CCPs. AT1R, Ang II type 1 receptor; A, agonist.

 
Several lines of evidence suggest that for efficient clathrin-mediated internalization, the endocytic proteins that are recruited into CCPs for initiating endocytosis must ultimately dissociate from the internalizing vesicle (29, 30). For instance, the dissociation of AP-2 from its internalized CCV is believed to be necessary to allow the recycling of the clathrin adapter for further rounds of CCP formation and receptor internalization. Thus, preventing the dissociation of AP-2 from ß-arrestin and/or AT1R would presumably affect the internalization of receptors. In support of this is our finding that AP-2 association with AT1R and ß-arrestin increases when c-Src expression was reduced in cells, and that under such condition the internalization of receptors was delayed. The accumulation of AP-2 to the receptor at the plasma membrane may ultimately impede the formation of subsequent CCPs, and slow down receptor internalization. Further studies will be necessary to determine the extent to which c-Src participate in the assembly and disassembly of CCPs and the internalization of other GPCRs.

In summary, our results provide a novel function for c-Src in the formation of endocytic complexes during the internalization of AT1R. They also provide another mechanism by which ß-arrestin, through the recruitment of signaling effectors like c-Src, can regulate the internalization of receptors through the clathrin pathway.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Ang II was purchased from Sigma (St. Louis, MO) [125I]-Ang II (1000 Ci/mmol) was obtained from Dr. Gaétan Guillemette (Université de Sherbrooke, Québec, Canada) and prepared as previously described (31). Antibodies against ß-adaptin and {alpha}-adaptin were from BD Transduction Laboratories (BD Biosciences, Palo Alto, CA), the c-Src (SRC2) and HA rabbit antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), the mouse anti-HA clone 12CA5 was from Roche Molecular Biochemicals (Indianapolis, IN), the FLAG antibody was from Sigma, and the c-Src antibody GD11 and the antiphosphotyrosine 4G10 were from Upstate Biotechnology (Lake Placid, NY). The rabbit polyclonal ß-arrestin antibody (A1CT) was kindly provided by Dr. Marc G. Caron (Duke University, Durham, NC).

Plasmids and Constructs
The HA-tagged c-Src and the HA-tagged c-Src-Y530F mutant were a gift from Dr. William E. Miller (University of Cincinnati, Cincinnati, OH). HA-tagged c-Src-K298R mutant was generated by PCR using the full-length HA-tagged c-Src in pcDNA3. A PCR fragment was amplified using a forward primer containing the mutation coding for the substitution K298R and a BamHI restriction site, and a reverse primer overlapping the KpnI site in c-Src. The fragment was digested with BamHI and KpnI, and replaced into c-Src cut with the same enzymes. Flag-tagged ß-arrestin R396A, and R396A/AAEA were generated by replacing an XhoI/XbaI fragment from the rat ß-arrestin2 wild type with a PCR product containing the mutations and the Flag sequence. The truncated Flag-tagged ß-arrestin2 (ßarr2-T372) was generated by PCR using a similar cassette replacement strategy to insert the Flag sequence after K372 in ß-arrestin. The p426-ADH-c-Src was generated by excising the avian c-Src mutant (c-Src-G2A,-Y416F,-Y527F) from the p413-Gal1 vector (kindly provided by Dr. Serge Lemay, McGill University, Québec, Canada) with BamHI and SalI, and cloning the fragment into p426-ADH (kindly provided by Dr. Bernard Turcotte, McGill University) using the same restriction sites. Green-fluorescent-tagged ß2-adaptin (GFP-ß2-adaptin) construct was described elsewhere (9). All constructs were analyzed by DNA sequencing (Service d’analyze et de synthèse d’acides nucléiques, Université Laval, Québec, Canada).

Cell Culture and Transfection
Human embryonic kidney cells (HEK 293) were grown in Eagle’s MEM (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS, Invitrogen Life Technologies) and gentamicin (100 µg/ml, Invitrogen Life Technologies). HEK 293 cells seeded in a 100-mm dish, at a density of 2.5 x 106 cells/dish, were transiently transfected using a conventional calcium phosphate coprecipitation method. One to 5 µg of DNA were mixed in a solution containing 125 mM CaCl2 in HEPES-buffered saline [25 mM HEPES (pH 7.4), 140 mM NaCl, 0.75 mM Na2HPO4] for 5 min, and the mixed solution was then added to cells. Green monkey kidney cells (COS-7) were grown in DMEM (Invitrogen Life Technologies) supplemented with 10% (vol/vol) heat-inactivated FBS and gentamicin (100 µg/ml). Transient transfections of COS-7 cells seeded in 60-mm dishes (at a density of 1 x 106 cells/dish) were performed using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer’s recommendations using a 1:3 ratio of DNA/Lipofectamine in Opti-MEM (Invitrogen Life Technologies). Twenty-four hours post transfection, HEK 293 and COS-7 cells were serum starved for 12 h in DMEM before performing the experiments. For vascular smooth muscle cells (VSMCs; kindly provided by Dr. Sylvain Meloche at the Université de Montréal, Québec, Canada), cells were grown in DMEM low glucose with 10% (vol/vol) heat-inactivated calf serum (Invitrogen Life Technologies) and gentamicin (100 µg/ml), and they were serum-starved in DMEM low glucose for 12 h before performing the experiments.

Immunoprecipitation and Western Blot Experiments
HEK 293 or COS-7 cells were left untreated or treated with Ang II (100 nM) at 37 C for the indicated period of time. After agonist stimulation, the reaction was stopped on ice by washing cells with ice-cold PBS. Cells were then solubilized in P-RIPA buffer (1 ml/100 mm dish) [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40 (vol/vol), 0.1% deoxycholate (wt/vol), 1 mM CaCl2] containing 0.1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 25 µg/ml leupeptine, 25 µg/ml aprotinin, and 1 mM pepstatin A for 30 min at 4 C. Cell lysates were centrifuged for 30 min at 21,000 x g and the supernatants were incubated with anti-Flag (30 µg/ml, Sigma) or c-Src antibody (3 µg/ml, clone GD11, Upstate) with 20 µl of a 50% slurry mixture of protein A/G Sepharose beads for 2 h at 4 C. Beads were washed three times with P-RIPA, and denatured in Laemmli buffer (2x) [250 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate (SDS) (wt/vol), 10% glycerol (vol/vol), 0.01% Bromophenol Blue (wt/vol), 5% ß-mercaptoethanol (vol/vol)]. Proteins were separated on a 10% SDS-PAGE before being transferred onto nitrocellulose membranes (Amersham Biosciences, Arlington Heights, IL). Membranes were then blocked with a solution of PBS (pH 7.4) containing 0.05% Tween 20 (vol/vol) (PBS-T) supplemented with 10% skin milk (wt/vol). Protein detection on membranes was assessed using either anti-c-Src antibody (1 µg/ml, clone GD11; or 0.2 µg/ml, SRC2), Flag antibody (2.5 µg/ml), HA antibody (0.2 µg/ml), phosphotyrosine antibody (0.2 µg/ml, clone 4G10), ß2-adaptin antibody (0.2 µg/ml, ß-adaptin), {alpha}-adaptin antibody (0.25 µg/ml), ß-arrestin antibody (diluted 1/5,000; A1CT) in PBS-T containing 1% BSA (fraction V; Sigma) (wt/vol) and 1% skin milk (wt/vol). After 1 h incubation with the different primary antibodies, membranes were washed three times in PBS-T before being incubated at room temperature with horseradish peroxidase-conjugated goat antimouse antibody (diluted 1/10,000; Sigma), or horseradish peroxidase-goat antirabbit antibody (diluted 1/10,000; Sigma) for 30 min. Immunoreactivity was visualized by enhanced chemiluminescence according to the manufacturer’s instructions (SuperSignal, Pierce, Rockford, IL).

Immunofluorescence Experiments
HEK 293 cells were grown on 22-mm2 coverslips in six-well plates (250,000 cells/well), and transfected with GFP-ß2-adaptin (0.5 µg) and HA-AT1R (1.5 µg). For AT1R immunodetection, cells were preincubated for 1 h at 14 C with a mouse anti-HA antibody (20 µg/ml, 12CA5), followed by incubation at 14 C for 1 h with a secondary goat antimouse antibody conjugated to AlexaFluor 568 (8 µg/ml, Molecular Probes, Eugene, OR). Labeled cells were stimulated at 37 C with 100 nM Ang II for 5 min, and fixed for 15 min at room temperature with a solution of 4% paraformaldehyde (wt/vol) in PBS. Coverslips were mounted with GelTol Aqueous Mounting Medium (IMMUNON, Thermo Shandon, Pittsburgh, PA). Confocal images of labeled cells were acquired with a Zeiss LSM-510 META laser-scanning microscope (Carl Zeiss, Jena, Germany) using a 60x oil immersion lens. GFP-ß2-adaptin and AlexaFluor 568-labeled receptor fluorescence was visualized using the multitrack mode with dual laser excitation (488 and 543 nm), and emission (BP 505–520 for GFP, and LP 560 for AlexaFluor 568) filter sets. Final figures were collated using Adobe Photoshop 7.0 and Illustrator 10.0 (Adobe, San Jose, CA).

Three-Hybrid Assay
Yeast two-hybrid MatchMaker System 2 (CLONTECH, BD Biosciences), was used with minor modifications to create a three-hybrid assay that allowed the detection of a ternary complex between ß-arrestin2, ß2-adaptin and c-Src. Constructs for GAL4-DBD ß-arrestin2 in pAS2-1 and GAL4-AD ß2-adaptin in pACT-2 were described elsewhere (9). PAS2-1-ß-arrestin2 and pACT-2-ß2-adaptin vectors were cotransformed with the empty vector p426-ADH or the p426-ADH-c-Src into the yeast strain PJ69-4a using the poly(ethylene-glycol) (PEG)/lithium acetate method (CLONTECH). Yeast colonies were grown on medium lacking tryptophan, leucine, and uracil for the selection of transformants expressing the bait vector (pAS2-1-ß-arrestin2), the prey vectors (pACT-2 or pACT-2-ß2-adaptin), and the bridge vector with or without c-Src (p426-ADH-c-Src or p426-ADH). Protein-protein interaction was assessed using the HIS3 auxotrophic complementation, and a lacZ reporter assay. For the ß-galactosidase assay, three colonies of each transformation were selected and grown on yeast extract peptone dextrose (YPD) medium at 30 C overnight. The next day, the yeast cultures were diluted to an A600 of 0.2 in synthetic dropout medium lacking tryptophan, leucine, and uracil, and grown for another 7 h at 30 C. Cultures were serially diluted at an A600 of 0.1, 0.002, and 0.004, and 10 µl of cell suspension were spotted onto medium lacking leucine, tryptophan, and uracil (–Leu/–Trp/–Ura) for selection of yeasts expressing the bait, the prey and the bridge vector, respectively, and selected onto medium lacking leucine, tryptophan, uracil, and histidine (–Leu/–Trp/–Ura/–His) for the detection of interactions between ß-arrestin2 and ß2-adaptin. Histidine complementation was assessed in presence of 2.5 mM 3-amino-1,2,4-triazole (3-AT, Sigma). ß-Galactosidase activity was measured using the ONPG assay (O-nitrophenyl ß-D-galactopyranoside, CLONTECH). Briefly, yeasts were grown on a selective medium to an A600 of 0.8–1.2, and cells from a 2-ml culture were collected by centrifugation. Pelleted cells were washed with cold Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 100 mM KCl, 1 mM MgSO4, and 30 mM ß-mercaptoethanol, at pH 7.0), and vigorously resuspended in 800 µl of Z-buffer containing 0.1% SDS (wt/vol) and 0.5% CHCl3 (vol/vol). Colorimetric reaction was started at 30 C by adding 160 µl of ONPG (4 mg/ml) to permeabilized cells. The reaction was stopped with 400 µl of Na2CO3 (1 M) when yellow coloration started to appear. The activity was measured at A420 and Miller units were calculated from the equation [Units = 1000 x (A420/ volume of cells assayed x time of run x A600)].

SiRNA and Antisense Experiments
All double-stranded siRNAs were synthesized using the Silencer siRNA kit (Ambion, Austin, TX) according to the manufacturer’s instructions. Twenty-one-oligomer corresponding to the sense and antisense sequences of the human c-Src mRNA were screened for unique sequence in the National Center for Biotechnology Information database by using the basic local alignment and search tool algorithm (accession no. BC011566). The target mRNA identified for c-Src is 5'-AAG CAC UAC AAG AUC CGC AAG-3', and corresponded to the position 607–628 (relative to the start codon) of c-Src. An extension that corresponded to the T7 promoter primer sequence (5'-CCT GTC TCT-3') was added at the 3' end of the sense and antisense sequence. Control siRNA sense and antisense oligonucleotides for GAPDH were provided in the kit.

For AT1R immunoprecipitation experiments, HEK 293 cells were seeded at a density of 400,000 cells/well in six-well plates, and transfected with Lipofectamine 2000. Briefly, pcDNA3.1 encoding the HA-AT1R (0.375 µg/well) was added with pcDNA3.1 (0.375 µg/well), siRNA-GAPDH (0.375 µg/well) or siRNA-c-Src (0.375 µg/well) and mixed with Lipofectamine 2000 (using a ratio of DNA/Lipofectamine of 1:2) in Opti-MEM. Complexes of DNA/Lipofectamine were added to cells in MEM without serum, and transfection was carried out for 12 h. The next day, media was replaced with complete MEM medium supplemented with 10% FBS (vol/vol) and gentamicin (100 µg/ml).

For immunofluorescence experiments, cells were seeded in six-well plates, at a density of 300,000 cells/well, and transfected with HA-AT1R (0.25 µg/well) and GFP-ß2-adaptin (0.125 µg/well) with or without siRNA-c-Src (0.375 µg/well) using the same DNA/Lipofectamine ratio as described above.

For AT1R internalization experiments, HEK 293 cells were seeded at a density of 70,000 cells/well in 24-well plates, and transfected with Lipofectamine 2000 as described above using the same DNA/Lipofectamine ratio. Briefly, pcDNA3.1 encoding the HA-AT1R (0.125 µg/well) was added with pcDNA3.1 (0.5 µg/well), siRNA-GAPDH (0.5 µg/well) or siRNA-c-Src (0.5 µg/well) and mixed with Lipofectamine 2000 in Opti-MEM. Transfection was carried out for 12 h before replacing the medium with complete MEM supplemented with 10% FBS (vol/vol) and gentamicin (100 µg/ml) and performing the experiment 72 h post transfection.

For experiments depleting c-Src in VSMCs, an antisense oligonucleotide strategy was used. Control scramble phosphorothioate (CTL-1, 5'-GTC TTA GCC GGG ATC CGC TA-3') and antisense oligonucleotides complementary to the sequence of the rat c-Src mRNA (AS-1, 5'-TT GCT CTT GTT GCT GCC CAT-3', accession number NM_031977) were designed and synthesized by Alpha DNA (Montreal, Québec, Canada). VSMCs were seeded at a density of 2 x 106 cells in 10-cm dishes and transfected with antisense DNAs using Lipofectamine 2000 as described previously. Briefly, c-Src AS-1 (1.2–6.0 µg, representing a final concentration of 50–250 nM in 4 ml) or CTL-1 (1.2–2.4 µg; 50–100 nM in 4 ml) were mixed using a ratio of oligonucleotide/Lipofectamine of 1:2 in Opti-MEM. Complexes were added to cells in DMEM (Invitrogen Life Technologies) without serum and transfection was carried out for 36 h before performing the experiments.

Internalization Assay
Receptor internalization was performed as described previously with minor modifications (31). Briefly, HEK 293 cells seeded in 24-well plates were transfected with HA-AT1R with or without siRNAs. Thirty-six hours post transfection cells were incubated at 37 C in DMEM containing 20 mM HEPES (pH 7.4), 0.1 mg/ml Bacitracin (Sigma) and 0.2% BSA (wt/vol), in presence of 0.11 nM of [125I]-Ang II for the indicated period of time. Incubation was stopped on ice by rapidly washing the cells three times with either ice-cold PBS to remove the unbound Ang II or ice-cold acid buffer [0.2 N acetic acid (pH 3.5), 150 mM NaCl] to remove both the unbound and the cell surface receptor-bound agonist. Cells were then solubilized in 0.5N NaOH, 0.05% SDS (wt/vol), and the radioligand content was evaluated by {gamma}-counting. Percent of receptor internalization was calculated from the ratio of acid-resistant binding over total binding (PBS wash). Data were analyzed by nonlinear regression using Prism4 (GraphPad Software, San Diego, CA).

Data Analysis
Intensity of the signals from Western blots was determined by densitometric analysis using Alpha Innotech Fluorochem imaging system (Packard Canberra, Montréal, Québec, Canada) and was represented as the mean ± SEM of at least three independent experiments. For densitometry analysis of Western blot and ß-galactosidase assay, data were analyzed statistically by one-way ANOVA followed by a Bonferroni posttest for multiple comparisons, or by one-tail t test for single comparison. Means were considered significantly different when P values were at least below 0.05.


    ACKNOWLEDGMENTS
 
We thank Drs. Stephen Ferguson and Audrey Claing for helpful discussion and comments. We are also grateful to the Claing lab for providing help with the siRNA.


    FOOTNOTES
 
This work was supported by a Canadian Institutes of Health Research Grant (MOP-49447) (to S.A.L.). M.S. holds a Fonds de Recherche en Santé du Quebec (FRSQ) fellowship. S.A.L is a recipient of a Canada Research Chair in Molecular Endocrinology.

First Published Online October 21, 2004

Abbreviations: AD, Activation domain; AP-1/2, clathrin adapter protein 1 and 2; Ang II, angiotensin II; AS-1, antisense DNA for c-Src; AT1R, angiotensin II type 1 receptor; ß2AR, ß2-adrenergic receptor; CCP, clathrin-coated pit; CCV, clathrin-coated vesicle; CTL-1, control antisense DNA; DBD, DNA binding domain; EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; HA, hemagglutinin; HEK, human embryonic kidney; siRNA, small interfering RNA; SDS, sodium dodecyl sulfate; VSMC, vascular smooth muscle cell.

Received for publication June 16, 2004. Accepted for publication October 15, 2004.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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