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J. Biol. Chem., Vol. 279, Issue 20, 21637-21642, May 14, 2004

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Distinct Roles for Src Tyrosine Kinase in ₂-Adrenergic Receptor Signaling to MAPK and in Receptor Internalization^*

Jianyun Huang, Yutong Sun, and Xin-Yun Huang

From the Department of Physiology, Cornell University Weill Medical College, New York, New York 10021

Received for publication, January 28, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
G protein-coupled receptors form the largest family of membrane receptors and transmit diverse ligand signals to modulate various cellular responses. After activation by their ligands, some of these G protein-coupled receptors are desensitized, internalized (endocytosed), and down-regulated (degraded). In HEK 293 cells, the G_s-coupled ₂-adrenergic receptor was postulated to initiate a second wave of signaling, such as the activation of the mitogen-activated protein kinase (MAPK) pathway after the receptor is internalized. The tyrosine kinase c-Src plays a critical role in these events. Here we used mouse embryonic fibroblast (MEF) cells deficient in Src family tyrosine kinases to examine the role of Src in ₂-adrenergic receptor signaling to the MAPK pathway and in receptor internalization. We found that in Src-deficient cells the ₂-adrenergic receptor could activate the MAPK pathway. However, the internalization of ₂-adrenergic receptors was blocked in Src-deficient MEF cells. Furthermore, we observed that in MEF cells deficient in -arrestin 2 the internalization of the ₂-adrenergic receptor was impaired, whereas the activation of the MAPK pathway by the ₂-adrenergic receptor was normal. Our data demonstrate that although Src and -arrestin 2 play essential roles in ₂-adrenergic receptor internalization, they are not required for the activation of the MAPK pathway by the ₂-adrenergic receptor. In other words, our finding suggests that receptor internalization is not required for ₂-adrenergic receptor signaling to the MAPK pathway in MEF cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
G protein-coupled receptors (GPCRs)¹ mediate transmembrane signaling for a large number of ligands including hormones, neurotransmitters, photons, odorants, pheromones, chemokines, and other stimuli (1-3). These receptors relay the signals to heterotrimeric G proteins, which directly modulate the activity of enzymes or other effector molecules. One of the prototypes of GPCRs is the ₂-adrenergic receptor (₂-AR) that transduces signals from catecholamines, norepinephrine, and epinephrine to the G_s protein, which in turn activates downstream effectors (2). After agonist activation, ₂-AR is phosphorylated by GPCR kinases with the subsequent binding of -arrestin 2 to the phosphorylated ₂-AR leading to the internalization (endocytosis) through clathrin-coated pits (4, 5).

GPCRs can relay ligand signals to various cellular signaling pathways. One of these cellular pathways is the mitogen-activated protein kinase (MAPK) pathway (6). The MAPK cascade, an evolutionarily conserved signaling module, stimulates numerous physiological responses including cell growth and differentiation (7). The pathway consists of a MAP kinase kinase kinase that phosphorylates and activates a MAP kinase kinase, which in turn phosphorylates the TXY activation loop of MAP kinase (8). The first characterized subfamily of MAP kinases, termed extracellular signal-regulated kinases (ERKs), and is activated by growth factor receptors, G protein-coupled receptors, and other types of receptors. The most common MAPK pathway involves Ras, Raf kinase (a MAP kinase kinase kinase), and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (a MAP kinase kinase) (9).

GPCRs seem to activate the ERK MAPK pathway by diverse signaling pathways depending on the receptors and the cell types. Some of these activation pathways from GPCRs to ERK MAPK involve tyrosine kinases (10-16). For example, previously we showed that Src family tyrosine kinases are essential for G_q signaling to ERK MAPK in DT40 chicken B lymphoma cells (11). In other GPCR signaling to ERK MAPK or in other cell types, tyrosine kinases might not be essential. We have shown that in S49 mouse T lymphoma cells, G_s signaling to the ERK MAPK pathway is independent of Src family tyrosine kinases. Instead, a protein kinase A-dependent pathway was used (17, 18). In HEK 293 cells, it was reported that isoproterenol stimulation of ₂-AR leading to the activation of ERK MAPK requires Src and receptor internalization (19).

Src family tyrosine kinases are a major group of cellular signal transducers (20). c-Src was the first identified protein tyrosine kinase (21, 22). These tyrosine kinases can be activated by various extracellular signals and modulate a variety of cellular functions including proliferation, survival, adhesion, and migration (20). GPCRs have been shown to stimulate tyrosine phosphorylation of cellular proteins (20). Various G protein-mediated physiological functions are sensitive to tyrosine kinase inhibitors. Many GPCRs are able to increase the activity of Src family tyrosine kinases (10-13, 15, 16, 23-27). Although the mechanism by which other G proteins activate Src family tyrosine kinases is not known, we have shown previously that G_s and G_i can directly interact with and activate Src (18, 28).

During our investigation of the role of tyrosine kinases in G protein signaling, we notice that in mouse embryonic fibroblast (MEF) cells deficient in Src family tyrosine kinases (SYF cells) a constitutively active mutant of G_s (G_sQ227L) is able to increase the kinase activity of ERK MAPK. Indeed stimulation of the endogenous G_s-coupled ₂-AR also activates ERK MAPK in SYF cells. However, the internalization of ₂-AR is blocked in SYF cells. Thus, these observations implicate that receptor internalization might not be necessary for the activation of the ERK MAPK pathway by ₂-AR. To confirm this, we used MEF cells deficient in -arrestin 2 because -arrestin 2 has been shown to be essential for ₂-AR internalization (29). We found that although -arrestin 2 deficiency impairs ₂-AR internalization, it does not affect ₂-AR signaling to the ERK MAPK pathway. Together, these data demonstrate that Src plays different roles in ₂-AR signaling to MAPK and in receptor internalization and that receptor internalization is not required for ₂-AR signaling to ERK MAPK in MEF cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Lines and Plasmid Constructs—The MEF cells deficient in Src family tyrosine kinases (SYF cells) were purchased from ATCC. The SYF/Src cells were established by stably transfecting human c-Src into SYF cells (28). The MEF cells deficient in either -arrestin 2 or deficient in -arrestin 1 and 2 were kindly provided by Dr. R. Lefkowitz (Duke University) (29). All plasmid cDNAs for human G proteins were purchased from Guthrie Research Institute. The cDNA for the human ₂-AR was kindly provided by Dr. C. Malbon (State University of New York at Stony Brook) and subcloned into the pcDNA3 vector. A GFP tag was inserted into the 3' end of ₂-AR.

ERK MAPK Assay—The p44/42 MAP kinase assay was performed using kits from Cell Signaling Technology as described previously (11). Whole cell lysates were prepared from MEF, SYF, SYF/Src, -arrestin ^--/-, or -arrestin 1^-/-2^-/- fibroblast cells. Cells were either treated or not with isoproterenol (10 µM) for 5 min. A monoclonal antibody to the phospho-p44/42 ERK MAPK (cross-linked to agarose beads) was added to immunoprecipitate the active ERK MAPK from cell lysates. Substrates (200 µM ATP and 2 µg glutathione S-transferase-Elk-1 fusion protein) were added, and the reaction was allowed to proceed at 30 °C for 30 min. After SDS-PAGE, the ERK MAPK activity (the phosphorylation of glutathione S-transferase-Elk-1 by ERK MAPK) was measured by Western blotting with an anti-phospho-Elk-1 antibody (Cell Signaling Technology).

Receptor Internalization—Cells were transiently transfected with a plasmid carrying a GFP-tagged human ₂-adrenergic receptor (pcDNA3-₂-AR-GFP). Twenty-four h after transfection, cells were split onto poly-D-lysine-coated glass coverslips. Forty-eight h after transfection, cells were treated with or without 10 µM isoproterenol at 37 °C for 30 min, rinsed quickly three times with phosphate-buffered saline, and further incubated in 3.7% formaldehyde for 20 min at room temperature. After being rinsed again three times in phosphate-buffered saline, the coverslips were mounted on a microscope slide with Vecta Shield mounting medium (Vector Laboratories) before imaging by fluorescence microscopy. Fluorescence microscopy was performed on a Zeiss Axiovert 35 microscope.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Src Family Tyrosine Kinases Are Not Required for G_sQ227L Stimulation of ERK MAPK in MEF Cells—To genetically test the role of Src family tyrosine kinases in G_s signaling to the ERK MAPK pathway, we investigated the stimulation of ERK MAPK by the constitutively active mutant of G_s (G_sQ227L) in Src family tyrosine kinase knock-out MEF cells. We used the SYF cells that were derived from Src, Yes, and Fyn triple knock-out mouse embryos (30). Because Src, Yes, and Fyn are the three ubiquitously expressed members of the Src family tyrosine kinases, no Src family tyrosine kinase activity was detected in these SYF cells (30). We found that transient expression of G_sQ227L in the control wild-type MEF cells led to increased activity of ERK MAPK (Fig. 1A). In SYF cells, expression of G_sQ227L also increased the ERK MAPK activity to a similar degree (Fig. 1B). These genetic data demonstrated that Src family tyrosine kinases are not required for G_sQ227L signaling to the ERK MAPK pathway. As controls, we also examined the requirement for Src family tyrosine kinases by some other G proteins signaling to ERK MAPK in MEF cells. As shown in Fig. 1C, transient expression of constitutively active mutants of G₁₂ (G₁₂Q231L), G₁₃ (G₁₃Q226L), G_q (G_qQ209L), G_i2 (G_i2Q205L), and G₁₂ led to the activation of ERK MAPK to different degrees. Interestingly, in SYF cells, except for G₁₃Q226L, the other G protein mutants (G_i2Q205L, G₁₂Q231L, G_qQ209L, and G₁₂) failed to stimulate ERK MAPK (Fig. 1D). These data suggest that G₁₃Q226L does not require Src family tyrosine kinases signaling to the ERK MAPK pathway in MEF cells, whereas G_i2Q205L, G₁₂Q231L, G_qQ209L, and G₁₂ require Src family tyrosine kinases for the activation of ERK MAPK in MEF cells. In this work, we focused on the G_s signaling to ERK MAPK. Thus, we did not examine further the Src-dependent signaling by other G proteins to the ERK MAPK pathway in MEF cells.

View larger version (28K): [in this window] [in a new window]   FIG. 1. Src family tyrosine kinases are not required for GsQ227L stimulation of ERK MAPK in MEF cells. A, top, transient expression of the constitutively active mutant GsQ227L increased the kinase activity of ERK MAPK in MEF cells. Whole cell lysates were prepared from MEF cells. Activated ERK MAPK proteins were immunoprecipitated from cell lysates by a monoclonal antibody against phospho-p44/42 ERK MAPK (cross-linked to agarose beads). The ERK MAPK activity was measured by the phosphorylation of substrate glutathione S-transferase-Elk-1 that was detected by Western blotting with an anti-phospho-Elk-1 antibody. Bottom, Western blotting with anti-ERK MAPK antibody showing that similar amounts of cell lysates were used in each lane. B, transient expression of GsQ227L increased the kinase activity of ERK MAPK in SYF cells. C, transient expression of constitutively active mutants of G12 (G12Q231L), G13 (G13Q226L), Gq (GqQ209L), Gi2 (Gi2Q205L), and G12 led to the activation of ERK MAPK in MEF cells. D, in SYF cells, G13Q226L (but not Gi2Q205L, G12Q231L, GqQ209L, and G12) stimulated the activity of ERK MAPK. Data are representative of four experiments.

View larger version (20K): [in this window] [in a new window]   FIG. 2. Src family tyrosine kinases are not required for 2-adrenergic receptor stimulation of ERK MAPK in MEF cells. A, isoproterenol (ISO) increased cAMP accumulation in MEF and SYF cells. Cells were treated with (or without) 10 µM isoproterenol for 5 min. Cellular levels of cAMP were measured with a cAMP assay kit from Assay Design Inc. B, isoproterenol increased the kinase activity of ERK MAPK in SYF and SYF/Src cells. Data are representative of three to five experiments.

View larger version (90K): [in this window] [in a new window]   FIG. 3. -Arrestin 2 is required for 2-adrenergic receptor internalization. A, fluorescent image of MEF cells transiently expressing 2-AR-GFP. MEF cells were transiently transfected with the plasmid carrying the GFP-tagged human 2-adrenergic receptor (pcDNA3-2-AR-GFP). B, fluorescence microscopy of MEF cells transiently expressing 2-AR-GFP after treatment with 10 µM isoproterenol (ISO) at 37 °C for 30 min. C, fluorescence microscopy of -arrestin 2-/- cells transiently expressing 2-AR-GFP. D, fluorescence microscopy of -arrestin --/- cells transiently expressing 2-AR-GFP after treatment with 10 µM isoproterenol at 37 °C for 30 min. Data are representative of three experiments.

View larger version (24K): [in this window] [in a new window]   FIG. 4. -Arrestin 2 is not essential for 2-adrenergic receptor signaling to ERK MAPK. MEF, -arrestin 2-/-, and -arrestin 1-/- 2-/- cells were treated with (or without) 10 µM isoproterenol (ISO) for 5 min. The ERK MAPK activity was measured. Data are representative of five experiments.

View larger version (95K): [in this window] [in a new window]   FIG. 5. Src is required for 2-adrenerigc receptor internalization. A, fluorescence microscopy of SYF cells transiently expressing 2-AR-GFP. B, fluorescence microscopy of SYF cells transiently expressing 2-AR-GFP after treatment with 10 µM isoproterenol (ISO) at 37 °C for 30 min. C, fluorescence microscopy of SYF/Src cells transiently expressing 2-AR-GFP. D, fluorescence microscopy of SYF/Src cells transiently expressing 2-AR-GFP after treating the cells with 10 µM isoproterenol at 37 °C for 30 min. Data are representative of five experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

However, there are disagreements on whether Src family tyrosine kinases are involved in ₂-AR signaling to the ERK MAPK pathway. In HEK 293 cells, -arrestin was reported to form a complex with Src and brought Src to the ₂-AR, leading to the receptor desensitization/internalization process, which initiates a second wave of signaling including the ERK MAPK pathway (19). On the other hand, we show here that in MEF cells Src is not essential for ₂-AR stimulation of ERK MAPK. Moreover, treatment of HEK 293 cells with Src family kinase inhibitors did not block G_s-coupled A_2A-adenosine receptor signaling to the ERK MAPK pathway (42). The different roles of Src in ₂-AR stimulation of ERK MAPK in these reports might reflect either the different cell types used or the methods used.

Interestingly, we noticed that the basal activity of ERK MAPK in SYF cells was consistently higher than in MEF cells and SYF/Src cells (Fig. 2B; also Fig. 1, A-D). This difference in basal activity of ERK MAPK is not caused by a difference in ERK MAPK protein expression levels in MEF and SYF cells. These data imply that the basal ERK MAPK activity in MEF cells was suppressed by c-Src. Inhibition of ERK by activated c-Src was reported recently in fibroblast cells (43). However, because MEF cells were serum-starved, c-Src was likely in the down-regulated (inactive) state. Thus, it seems that the down-regulated c-Src has an unexpected function here. This might be reminiscent of the MAPK Kss1 in the yeast Saccharomyces cerevisiae. Kss1 in its inactive form is a potent negative regulator of invasive growth (44, 45). The nature of this potential negative regulation of basal ERK MAPK by c-Src needs further investigation.

Role of Src Family Tyrosine Kinases in ₂-Adrenergic Receptor Internalization—For ₂-AR internalization, ligand stimulation of ₂-AR leads to the activation of G protein-coupled receptor kinases and the phosphorylation of some C-terminal Ser/Thr residues of ₂-AR by G protein-coupled receptor kinases. This phosphorylation in turn promotes the association of ₂-AR with -arrestin 2 and subsequent internalization (4, 5). The essential role of -arrestin 2 in ₂-AR internalization was confirmed in MEF cells from -arrestin 2 knock-out mice (29). -Arrestins can interact directly with clathrin (46), which could promote receptor internalization through clathrin-coated pits (4, 5).

Following the above ₂-AR internalization scheme, it was surprising to see the inhibition of ₂-AR internalization in SYF cells. Our data strongly indicate that Src family tyrosine kinases are involved in the ₂-AR internalization process. There was some suggestive evidence that Src family tyrosine kinases might act upstream of G protein-coupled receptor kinases in ₂-AR internalization. First, overexpression of a dominant-negative mutant of c-Src, treatment with PP2 inhibitor, and antisense oligodeoxynucleotides in human epidermoid A431 carcinoma cells inhibited ligand-induced ₂-AR internalization (47). It was proposed that ligand stimulation of ₂-AR leads to the phosphorylation of tyrosine residue 350 at the C-terminal tail of the ₂-AR by a yet to be identified tyrosine kinase. Src then uses its Src homology 2 domain to bind this phosphorylated tyrosine 350. This association to the ₂-AR also activates Src (47). Second, it was reported that Src might phosphorylate and activate G protein-coupled receptor kinases (48). The detailed mechanism by which Src family tyrosine kinases participate in the ₂-AR internalization needs further investigation (49). Nevertheless, our data clearly show that Src is essential for ₂-AR internalization in MEF cells.

Role of Receptor Internalization in ₂-Adrenergic Receptor Signaling to ERK MAPK—Previous studies that used a dominant-negative dynamin mutant and/or mutant arrestin proteins to examine the potential role of receptor internalization in the ₂-AR signaling to ERK MAPK yielded inconsistent results. In HEK 293 cells and COS-7 cells, although some researchers reported that receptor internalization was required for ₂-AR initiated activation of the ERK MAPK pathway (19, 50, 51), others reported no requirement for receptor internalization in ₂-AR signaling to ERK MAPK (40, 52). Our genetic data clearly demonstrate that the receptor internalization is not essential for ₂-AR signaling to ERK MAPK in MEF cells.

For GPCRs as a whole, whether the receptor internalization is needed for the activation of the ERK MAPK pathway remains to be fully clarified. Some researchers suggested that receptor internalization seems to be needed for activation of the ERK MAPK pathway by some GPCRs (51, 53, 54), whereas others believed that receptor internalization is not essential for ERK MAPK activation (52, 55-58). Quite a few reports used dominant-negative mutants of dynamin to evaluate the role of receptor endocytosis in ERK MAPK activation, but caution needs to be exercised when interpreting the results because it has now been reported that dynamin is required for the activation of ERK MAPK by its upstream kinase MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) in cells (59). It might be helpful to use genetic tools such as loss-of-function mutants or deletions of specific signaling components to demonstrate whether receptor internalization is indeed essential for the signaling of a particular GPCR to ERK MAPK.

In summary, we have shown that Src family tyrosine kinases are not required for G_sQ227L stimulation of ERK MAPK in MEF cells. Ligand stimulation of the endogenous G_s-coupled ₂-AR also activates ERK MAPK in SYF cells, whereas the internalization of ₂-AR is blocked in SYF cells. Furthermore, we found that although the -arrestin 2 deficiency impairs ₂-AR internalization, it has no effect on ₂-AR signaling to the ERK MAPK pathway. Therefore, Src and -arrestin 2 play different roles in ₂-AR signaling to ERK MAPK and in receptor internalization, and receptor internalization is not required for ₂-AR signaling to ERK MAPK in MEF cells.

	FOOTNOTES

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

These two authors contributed equally to this work.

An American Heart Association established investigator, Irma T. Hirschl Trust career scientist, and Charles H. Leach Foundation research scholar. To whom correspondence should be addressed. Tel.: 212-746-6362; Fax: 212-746-8690; E-mail: xyhuang{at}med.cornell.edu.

¹ The abbreviations used are: GPCR, G protein-coupled receptor; ₂-AR, ₂-adrenergic receptor; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEF, mouse embryonic fibroblast; GFP, green fluorescent protein.

	ACKNOWLEDGMENTS

 
We thank Dr. R. Lefkowitz for the MEF cells deficient in -arrestin proteins. We thank T. Maack, D. McGarrigle, and X. Zhou for reading the manuscript.

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