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J. Biol. Chem., Vol. 275, Issue 49, 38131-38134, December 8, 2000

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ACCELERATED PUBLICATION
Direct Binding of Activated c-Src to the ₃-Adrenergic Receptor Is Required for MAP Kinase Activation^*

Wenhong Cao, Louis M. Luttrell, Alexander V. Medvedev, Kristen L. Pierce^§, Kiefer W. Daniel, Tonya M. Dixon, Robert J. Lefkowitz^§, and Sheila Collins^¶

From the Departments of Psychiatry and Behavioral Sciences, ^¶ Pharmacology, and  Medicine and the ^§ Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, August 30, 2000, and in revised form, September 20, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Both ₂- and ₃-adrenergic receptors (ARs) are able to activate the extracellular signal-regulated kinase (ERK) pathway. We previously showed that c-Src is required for ERK activation by ₂AR and that it is recruited to activated ₂AR through binding of the Src homology 3 (SH3) domain to proline-rich regions of the adapter protein -arrestin1. Despite the absence of sites for phosphorylation and -arrestin binding, ERK activation by ₃AR still requires c-Src. Agonist activation of ₂AR, but not ₃AR, led to redistribution of green fluorescent protein-tagged -arrestin to the plasma membrane. In -arrestin-deficient COS-7 cells, -agonist-dependent co-precipitation of c-Src with the ₂AR required exogenous -arrestin, but activated ₃AR co-precipitated c-Src in the absence or presence of -arrestin. ERK activation and Src co-precipitation with ₃AR also occurred in adipocytes in an agonist-dependent and pertussis toxin-sensitive manner. Protein interaction studies show that the ₃AR interacts directly with the SH3 domain of Src through proline-rich motifs (PXXP) in the third intracellular loop and the carboxyl terminus. ERK activation and Src co-precipitation were abolished in cells expressing point mutations in these PXXP motifs. Together, these data describe a novel mechanism of ERK activation by a G protein-coupled receptor in which the intracellular domains directly recruit c-Src.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

During the past several years, transmembrane signaling traffic through G protein-coupled receptors (GPCRs)¹ has grown from the classic G protein effectors such as adenylyl cyclase and phospholipases to include novel mechanisms for activation of mitogen-activated protein (MAP) kinase cascades. These signaling systems typically involve receptor and non-receptor tyrosine kinases as scaffolds and intermediaries (1-6). An example of this flexibility in GPCR signaling includes the ₂-adrenergic receptor (₂AR). Although this receptor is classically known to couple to Gs and stimulate adenylyl cyclase, it can also activate the ERK1/2 MAP kinase pathway (7, 8). In some cell types, the ₂AR activates ERK through its coupling to a PTX-sensitive G_i protein and subsequent Ras-dependent MAP kinase activation (7, 9), whereas in other systems this occurs in a PTX-independent and a G_s- and cAMP-dependent process (10, 11) through the activation of Rap1 (11).

In exploring the mechanisms of ₂AR-stimulated MAP kinase activation, we have found that some of the same signaling molecules required for receptor desensitization can also be intimately involved in the activation of the MAP kinase cascade. Following agonist activation, most GPCRs are phosphorylated by GPCR kinases (GRKs), with subsequent binding of -arrestin to the phosphorylated receptor serving to interdict G protein coupling and signal transduction (5, 12, 13). However, in addition to its role in desensitization, -arrestin can also participate in the events leading to MAP kinase activation. Binding of -arrestin1 to the agonist-activated ₂AR rapidly recruits c-Src to the receptor (12, 14). This recruitment appears to be mediated by an interaction between the amino-terminal proline-rich region of -arrestin1 and the SH3 domain of c-Src (13, 15).

The ₃AR is a member of the AR subfamily of GPCRs that is expressed predominantly in adipocytes. Because selective ₃AR agonists have been shown to prevent or even reverse obesity and diabetes in various animal models (16-18), increased attention has been focused upon the molecular and physiological regulation of this receptor as a therapeutic target (19). Early studies of -adrenergic stimulation of adenylyl cyclase in adipocytes by Rodbell and colleagues (20) indicated the presence of a PTX-sensitive component. In examining this issue, we showed that this effect is due to the presence of the adipocyte-specific ₃AR and its ability to simultaneously couple to both G_s and G_i, leading to the activation of the cAMP-dependent protein kinase A and ERK1/2 pathways, respectively (9). Because GRK-mediated phosphorylation is necessary for -arrestin binding (reviewed in Ref. 21), but the ₃AR lacks sites for phosphorylation (22), we concluded that the ₃AR must employ a novel mechanism of ERK activation. Here, we demonstrate that conserved proline-rich motifs in the third intracellular loop and carboxyl terminus of the ₃AR directly recruit c-Src in a ₃AR agonist- and PTX-sensitive manner. This interaction occurs specifically through the SH3 domain of c-Src. Our findings establish a new mechanism whereby some GPCRs can acquire ligand-induced tyrosine kinase activity by means of direct recruitment of Src kinases.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Chemicals and Construction of Plasmids-- -The ₃AR agonist CL316,243 was a gift from American Cyanamid Co. (Pearl River, NY). L-()-Isoproteronol and propranolol were from Sigma and selective c-Src inhibitor PP2 from Calbiochem. For plasmids Xa-2-Loop3 (Wt), Xa-2-Tail (Wt), Xa-2-MutL1 (L1), and Xa-2-MutT1 (T1), the wild-type or mutated third intracellular domain (amino acids 222-289) or the carboxyl terminus (amino acids 344-400) of mouse ₃AR were cloned into PinPoint Xa-2 vector at HindIII and BglII sites under the control of the SV40 promoter. All plasmid constructs were verified by sequencing. GST fusion proteins of c-Src containing either the SH3 and SH2 domains (GST-SH3/SH2) or the SH2 domain alone (GST-SH2) were prepared as described previously (23).

Cell Culture and Transfections and Signal Transduction Assays-- C3H10T1/2 (T1/2) preadipocytes and COS-7 cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum in 6-well dishes as detailed (13, 24). Cells were transfected with HA-₃AR DNA (2 µg for T1/2 cells; 1 µg for COS-7 cells) and 5 µl of LipofectAMINE (Life Technologies, Inc.). For T1/2, on day 2 cells were induced to differentiate (1 µM rosiglitazone, 0.1 µM LGD1069, 200 nM insulin). Prior to each analysis, the density of receptor per cell was assessed by fluorescence-activated cell sorting. MAP kinase assays were performed as previously detailed (9) in cells that were serum-deprived for 24 h. Where indicated, some cells were treated with PTX (100 ng/ml, 16 h) propranolol (0.1 µM, 5 min), CL316,243 (10 µM), or isoproterenol (10 µM, 5 min). cAMP levels in whole cells were measured as described previously (9).

Confocal Fluorescence Microscopy-- Confocal microscopy was performed on a Zeiss LSM510 laser scanning microscope using a Zeiss 63X 1.4 numerical aperture water immersion lens, and fluorescent signals were collected (14). Three independent experiments were performed, and 10 or more fields/sample were analyzed in each experiment.

Protein-Protein Interaction and Co-precipitation Assays-- The biotinylated fusion proteins for the wild-type or mutated Xa-2-₃AR Loop3 or Xa-2-₃AR Tail were expressed and purified (Promega). For in vitro binding, 2 µg of purified biotinylated fusion protein was mixed with 4 µg of GST-Src-SH3/2 or GST-Src-SH2 fusion proteins and incubated in phosphate-buffered saline containing 1% bovine serum albumin, 5% glycerol, and 0.1% Nonidet P-40. After 16 h at 4 °C, the reactions were terminated by washing the immobilized complexes with 30 volumes of ice-cold phosphate-buffered saline containing 5% glycerol and 0.1% Nonidet P-40. The supernatant was removed, and 50 µl of 2× SDS-polyacrylamide gel electrophoresis sample buffer was added to each reaction. The biotinylated proteins were resolved by 4-20% SDS-acrylamide gradient gel electrophoresis (Novagen), transferred to nitrocellulose membranes, and identified by staining with streptavidin-conjugated alkaline phosphatase. Immunoprecipitations of HA-tagged ₂AR and ₃AR from intact cells and immunoblotting for co-precipitated proteins were performed as described previously (25).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Activation of the Ras-dependent ERK cascade by many GPCRs requires Src kinase activity (13, 25). For the ₂AR, ERK activation depends on the delivery of -arrestin-bound c-Src to the receptor (13). However, it is unclear whether other GPCRs utilize this same mechanism. As shown in Fig. 1, ₃AR-mediated ERK activation similarly requires Src kinase activity, as demonstrated by its concentration-dependent sensitivity to the Src-specific tyrosine kinase inhibitor, PP2. Complete inhibition was achieved within the range of 1 to 5 µM; a concentration previously established to selectively inhibit Src kinase (8, 26). Inhibition of ₃AR-mediated ERK activation was also observed when the C-terminal Src kinase was co-expressed with ₃AR (data not shown). Because GRK-mediated phosphorylation of receptors is necessary for -arrestin binding (21), but the ₃AR is not phosphorylated by GRKs, we hypothesized that agonist stimulation of ₃AR would not lead to -arrestin binding. This hypothesis is confirmed as illustrated in Fig. 2, which compares the effects of agonist stimulation on the cellular distribution of a chimeric -arrestin 2-GFP in HEK-293 cells expressing either the human ₂AR or the mouse ₃AR. Isoprenaline (10 µM) stimulation of the ₂AR promotes the rapid translocation of -arrestin 2-GFP from a diffuse cytosolic distribution to the plasma membrane where it aggregates with the receptor in membrane-associated puncta (14). In contrast, stimulation of cells expressing the mouse ₃AR with the selective ₃AR agonist CL316,243 (5 µM) fails to induce -arrestin 2-GFP translocation. Thus, although ₃AR-stimulated ERK activation is Src-dependent, similar to the ₂AR, the ₃AR response occurs without the formation of complexes between ₃AR and -arrestin.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   The c-Src tyrosine kinase inhibitor PP2 inhibits the activation of ERK1/2 by 3AR receptors. A, COS-7 cells mock transfected (NT) or expressing 3AR were pretreated with 0.1 µM propranolol and indicated concentrations of PP2 for 15 min prior to stimulation with 10 µM CL316,243 for 5 min. Cell lysates were prepared and analyzed by immunoblotting with antisera for ERK1/2 as described previously (9). B, Quantification of the results described in A shows the mean ± S.E. for 3 independent experiments.

View larger version (71K): [in this window] [in a new window]   Fig. 2.   Stimulation of 2AR, but not 3AR, results in recruitment of -arrestin 2-GFP to the plasma membrane. Cells expressing HA epitope-tagged 2AR (upper panel) or 3AR (lower panel) and -arrestin 2-GFP chimera were stimulated for 10 min with 10 µM isoproterenol or 5 µM CL316,243, respectively. The subcellular distribution of -arrestin 2-GFP before (left panels) and after (right panels) agonist stimulation was determined by laser confocal immunofluorescence microscopy (13). Frames shown are from 1 of 3 independent experiments.

We previously showed that c-Src interacts with proline-containing motifs in the -arrestin amino terminus and the SH3 domain of c-Src, although the c-Src catalytic domain also contributes significantly to this binding (13). Interestingly, although the ₃AR does not recruit -arrestin, all species homologues of this receptor contain highly conserved proline residues in both the third intracellular domain and the carboxyl terminus that are completely absent from the ₂AR. Two of these proline clusters in each domain contain the sequence PXXP, which represents the minimal consensus motif for SH3 domain binding (27-29). We therefore tested the hypothesis that these proline-rich motifs within the ₃AR might directly recruit SH3 domain-containing proteins to the receptor, obviating the need for -arrestin to function as an adapter protein.

First, we determined whether ₃AR could directly recruit Src kinases to activate the ERK pathway in the absence or presence of over-expressed -arrestin in COS-7 cells, which express little endogenous -arrestin (30). As shown in Fig. 3 (lanes 1-3), agonist treatment resulted in the detectable co-precipitation of c-Src with the ₂AR only in the presence of co-expressed -arrestin. As expected from earlier studies (13), the provision of -arrestin dramatically enhanced ₂AR-mediated ERK phosphorylation under these conditions. In contrast, the expression of -arrestin had no effect on responses mediated by the ₃AR. Robust ₃AR agonist-dependent co-precipitation of c-Src and ERK1/2 activation was observed, which did not require the presence of -arrestin (lanes 4-6). Src that co-precipitated with ₂AR and ₃AR was also in its activated (dephosphorylated) state.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   -Arrestin is required for the association of c-Src with 2AR but not with 3AR. COS-7 cells were transfected with c-Src and HA epitope-tagged 2AR or 3AR with or without -arrestin. Transfected cells were treated with 5 µM isoprenaline (ISO) for 2 min or 5 µM CL316,243 for 5 min, as indicated. Immunoprecipitation (IP) of HA-tagged receptors was performed, and co-precipitated c-Src was detected by immunoblotting with specific antisera for total c-Src and activated (Tyr530 dephosphorylated) Src, as described previously (13). Aliquots of whole cell lysates were resolved in parallel and immunoblotted for ERK1/2 (9). The data shown are from 1 of 3 independent experiments.

Second, because the physiological site of expression of the ₃AR is the adipocyte, a key question is whether the ₃AR directly recruits Src kinase in adipocytes as observed in COS-7 cells. We performed similar co-precipitation experiments in the mouse white adipocyte cell line, C3H10T1/2 (31). As shown in Fig. 4A, by day 4 of differentiation, C3H10T1/2 cells express the adipocyte-specific genes ₃AR and the fatty acid-binding protein aP2 (32, 33). Fig. 4B shows that the ₃AR-selective agonist CL316,243 is capable of triggering ERK activation in both nontransfected (NT) cells (via the endogenous ₃AR), and in the HA-m₃AR transfected cells, but ERK activation was abolished by inactivation of G_i with PTX. Fig. 4C shows that, as observed in COS-7 cells, Src kinase co-precipitates with the HA-₃AR in C3H10T1/2 adipocytes, and this interaction is both agonist- and G_i-dependent.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   3AR binds to c-Src in a Gi protein- and agonist-dependent manner in differentiated C3H10T1/2 adipocytes. C3H10T1/2 cells were transfected with HA-3AR and differentiated as described under "Experimental Procedures." Immunoprecipitation assays (IP) were performed with anti-HA antibody. A, expression of aP2 and 3AR mRNA as a function of differentiation on the indicated days. Levels of aP2 are maximal by day 4. Cyclophilin RNA (Cyclo) is a control used in Northern blotting (17, 40). B, the level of phosphorylated ERK1/2 (ERK1/2-P) in cell lysates and quantification of 3 independent experiments (mean ± S.D). C, the level of c-Src co-precipitated with 3AR and quantification of 3 independent experiments (mean ± S.D.).

These results taken together indicate that the ₃AR can mediate the -arrestin-independent recruitment of c-Src. To address whether binding between proline-rich motifs in the ₃AR and the c-Src SH3 domain might be responsible for this interaction, we first tested whether peptides derived from these regions of the ₃AR, as shown in Fig. 5A, would bind to GST-Src fusion proteins in vitro. As shown in Fig. 5B, biotinylated fusion proteins derived from both the third intracellular domain (Loop3) and the carboxyl terminus (Tail) of the wild-type ₃AR bound to the GST-Src SH3/SH2 but not to the GST-Src SH2 fusion protein. There was no interaction with GST alone. Consistent with an SH3 domain-mediated interaction, mutants of the ₃AR Loop3 (L1) and Tail (T1) peptides in which Ser was substituted for Pro in the PXXP motifs were no longer able to interact with the GST-Src SH3/SH2 peptide. In addition, we found that the wild-type ₃AR Loop3 and Tail peptides could precipitate endogenous c-Src and Grb2 from whole cell lysates of HEK-293 cells (not shown). Collectively, these data suggest that proline-rich motifs in both Loop3 and the Tail of the ₃AR possess the capacity to bind SH3 domains.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Proline-rich motifs in 3AR bind c-Src in a SH3-dependent manner. A, sequences of proline-rich motifs in the third intracellular domain (Loop3) and the carboxyl terminus (Tail) of the mouse 3AR. Mutations that eliminate the PXXP motifs in Loop3 (L1 and L2) and Tail (T1) are shown. B, binding of 3AR Loop3 and Tail proteins to GST-Src fusion proteins containing both the SH3 and SH2 domains (amino acids 87-249) or the SH2 domain alone (amino acids 144-249) were detected by phosphatase-coupled streptavidin.

To assess the functional role of the ₃AR proline-rich motifs in Src-dependent activation of the ERK cascade, mutant ₃ARs were constructed in which these motifs were disrupted by site-directed mutagenesis. Mouse ₃ARs containing the L1, L2 (a deletion of Loop3 amino acids Ser-242 to Pro-266), or T1 mutations were expressed in COS-7 cells and assayed for the ability to co-precipitate endogenous c-Src and to induce ERK1/2 phosphorylation and cAMP production. As shown in Fig. 6, mutation or deletion of the PXXP motifs in either the third intracellular loop or the carboxyl terminus resulted in a striking dissociation of ₃AR-mediated c-Src binding and ERK activation from ₃AR-mediated stimulation of adenylyl cyclase. Fig. 6A shows that endogenous c-Src co-precipitated with wild-type ₃AR in an agonist-dependent manner, whereas co-precipitation of c-Src with each of the mutant receptors was markedly impaired. Similar effects were observed for ₃AR-mediated ERK1/2 activation, which was abolished by the L1, L2, and T1 mutations (Fig. 6C). In contrast, ₃AR-mediated production of cAMP was completely unaffected by disruption of the PXXP motifs (Fig. 6E).

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Proline-rich motifs of 3AR are required for c-Src binding and stimulation of ERK1/2. A, co-precipitation of endogenous c-Src in COS-7 cells transfected with expression constructs for HA-tagged wild-type (WT) or mutant (L1, L2, or T1) mouse 3AR. Immunoprecipitates (IP) were prepared using anti-HA-agarose and probed with anti-Src antibody. B, quantification of c-Src co-precipitation. The results are the mean ± S.E. for 3 independent experiments, normalized to nonstimulated cells transfected with each receptor construct. C, level of phospho-ERK1/2 in the cell lysates. D, quantification of phospho-ERK1/2 activated by CL316,243 in cells transfected with each receptor/mutant. Results are the mean ± S.E. for 3 independent experiments, normalized to non-stimulated cells. E, cells expressing wild-type or mutant 3ARs were analyzed for basal and CL316,243-stimulated cAMP production as previously described (9).

These data suggest that the ability of the ₃AR to mediate Src-dependent activation of the ERK cascade, but not its ability to interact with G_s protein and stimulate the cAMP pathway, is dependent upon the integrity of the PXXP motifs in the third intracellular domain and the carboxyl terminus of the receptor. Each of these motifs is sufficient to bind to Src-GST SH3 domains in vitro, and site-directed mutagenesis of either motif prevents the agonist-dependent formation of complexes between the ₃AR and c-Src when the mutant receptors are expressed in intact cells. The apparent necessity for intact PXXP motifs in both the third intracellular loop and the carboxyl terminus of the ₃AR for agonist-dependent Src co-precipitation and functional ERK activation is not clear, but it implies that a novel multimeric complex containing at least activated c-Src and two domains of the ₃AR is formed on the receptor itself to trigger this signaling cascade. However, the exact nature of this complex is unknown, and the stoichiometry of the receptor-Src interaction will require further mutagenesis and structural analysis.

The distinct strategies employed by the ₂AR and ₃AR to recruit Src kinases illustrate the flexibility that characterizes the mechanisms of G protein coupling, agonist-induced receptor sequestration, and activation of tyrosine protein kinases by heptahelical receptors. In the case of the ₂AR, G protein coupling and ERK activation occur sequentially. Src recruitment requires -arrestin binding, an event that simultaneously terminates receptor-G protein coupling and triggers removal of the receptor from the cell surface. In contrast, the ₃AR is expressed in adipocytes where it is stimulated by norepinephrine in response to the requirement for fuel mobilization and heat generation (34). Given its physiologic role, there is little apparent need for ₃AR desensitization, particularly in times of chronic stimulation such as exposure to cold. There is increasing evidence that the coincident activation of the cAMP-dependent protein kinase A and MAP kinase pathways could have important consequences for energy balance (35-38), given the potent and robust effects of ₃AR agonists in vivo (17, 39). Inclusion of SH3 domain binding motifs within the intracellular domains of the receptor incorporates the adapter protein role of -arrestin within the receptor, thus allowing Src recruitment and ERK activation to proceed independently of receptor sequestration.

	ACKNOWLEDGEMENTS

We thank Dr. Bruce Spiegelman for the cDNA encoding aP2, Drs. Larry Barak and Marc Caron for the -arrestin 2-GFP clone, and Dr. William C. Wetsel for reading the manuscript.

	FOOTNOTES

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

To whom correspondence should be addressed: Duke University Medical Ctr., Box 3557, Durham, NC 27710. Tel.: 919-684-8991; Fax: 919-684-3071; E-mail: colli008@mc.duke.edu.

Published, JBC Papers in Press, September 29, 2000, DOI 10.1074/jbc.C000592200

	ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; MAP, mitogen-activated protein; ₂/₃AR, ₂/₃-adrenergic receptor; ERK, extracellular signal-regulated kinase; PTX, pertussis toxin; GRK, GPCR kinase; SH3, Src homology 3; GST, glutathione S-transferase; GFP, green fluorescent protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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				D. S. Hutchinson, M. Sato, B. A. Evans, A. Christopoulos, and R. J. Summers Evidence for Pleiotropic Signaling at the Mouse {beta}3-Adrenoceptor Revealed by SR59230A [3-(2-Ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol Oxalate] J. Pharmacol. Exp. Ther., March 1, 2005; 312(3): 1064 - 1074. [Abstract] [Full Text] [PDF]

				C. J. Hupfeld, J. L. Resnik, S. Ugi, and J. M. Olefsky Insulin-induced {beta}-Arrestin1 Ser-412 Phosphorylation Is a Mechanism for Desensitization of ERK Activation by G{alpha}i-coupled Receptors J. Biol. Chem., January 14, 2005; 280(2): 1016 - 1023. [Abstract] [Full Text] [PDF]