Clathrin-mediated endocytosis of m3 muscarinic receptors. Roles for Gβγ and tubulin

Receptors as well as some G protein subunits internalize after agonist stimulation. It is not clear whether Gαq or Gβγ undergo such regulated translocation. Recent studies demonstrate that m3 muscarinic receptor activation in SK-N-SH neuroblastoma cells causes recruitment of tubulin to the plasma membrane. This subsequently transactivates Gαq and activates phospholipase Cβ1. Interaction of tubulin-GDP with Gβγ at the offset of phospholipase Cβ1 signaling appears involved in translocation of tubulin and Gβγ to vesicle-like structures in the cytosol (Popova, J. S., and Rasenick, M. M. (2003) J. Biol. Chem. 278, 34299–34308). The relationship of this internalization to the clathrin-mediated endocytosis of the activated m3 muscarinic receptors or Gαq involvement in this process has not been clarified. To test this, SK-N-SH cells were treated with carbachol, and localization of Gαq, Gβγ, tubulin, clathrin, and m3 receptors were analyzed by both cellular imaging and biochemical techniques. Upon agonist stimulation both tubulin and clathrin translocated to the plasma membrane and co-localized with receptors, Gαq and Gβγ. Fifteen minutes later receptors, Gβγ and tubulin, but not Gαq, internalized with the clathrin-coated vesicles. Coimmunoprecipitation of m3 receptors with Gβγ, tubulin, and clathrin from the cytosol of carbachol-treated cells was readily observed. These data suggested that Gβγ subunits might organize the formation of a multiprotein complex linking m3 receptors to tubulin since they interacted with both proteins. Such protein assemblies might explain the dynamin-dependent but β-arrestin-independent endocytosis of m3 muscarinic receptors since tubulin interaction with dynamin might guide or insert the complex into clathrin-coated pits. This novel mechanism of internalization might prove important for other β-arrestin-independent endocytic pathways. It also suggests cross-regulation between G protein-mediated signaling and the dynamics of the microtubule cytoskeleton.

Tubulin is a structural protein that builds the microtubule network of the cell. Tubulin also regulates adenylyl cyclase and phospholipase C␤ 1 (PLC␤ 1 ) 1 signaling through GTP transacti-vation of the ␣ subunits of the G proteins G s , G i , and G q (1)(2)(3)(4). G␣ i1 and G␤ 1 ␥ 2 in turn reciprocally regulate microtubule assembly through their interaction with tubulin (5,6). This crossregulation between intracellular signaling and microtubule dynamics may be controlled by extracellular signals and perhaps still unknown intracellular events.
Tubulin-GTP transactivates G␣ q and activates PLC␤ 1 signaling in response to carbachol stimulation of SK-N-SH neuroblastoma cells (4,7). After PLC␤ 1 activation membrane-associated tubulin-GDP appears to interact with G␤␥, and the two proteins internalize in vesicle-like structures in the cytosol (8). The endocytic mechanism involved in G␤␥/tubulin internalization has not been revealed.
Many activated receptors sequester in clathrin-coated pits and undergo endocytosis after GRK phosphorylation and subsequent binding of ␤-arrestin at these phosphorylated sites. ␤-Arrestin binds to the heavy chain of clathrin and the AP2 adaptor protein and, thus, translocates activated receptors to the clathrin-coated pits (9,10). However, internalization of some receptors with the clathrin-coated vesicles appears not to depend upon ␤-arrestin (11)(12)(13)(14)(15)(16)(17)(18). It has been reported that, although m 1 and m 3 muscarinic receptors are phosphorylated by GRK (19 -21) and internalize through clathrin-mediated endocytosis, their internalization involves dynamin but not ␤-arrestin (11). This ␤-arrestin-independent internalization is not well understood. However, G␤␥ binding to the third intracellular loop of m 3 muscarinic receptors may be instrumental in their internalization (22).
G␤␥ subunits are multifunctional complexes that regulate multiple events. They control activation of adenylyl cyclases and PLC␤ isozymes, ion channels, and kinases (for review, see Refs. 23 and 24). They also target cytosolic GRKs and phosphoinositide 3-kinases as well as PLC␤ 1 to the membrane of the cell (25)(26)(27). G␣ s , G␣ q , and G␣ z attachment to the plasma membrane requires G␤␥ subunits (28 -30). G␤␥ also assists activated receptors to pry open the G␣-GDP binding pocket and release the guanine nucleotide (30). G␤␥ is also involved in dissociation of tubulin from the plasma membrane at the offset of PLC␤ 1 signaling after m 3 muscarinic receptor activation (8). Because m 3 receptors appear to require dynamin but not ␤-arrestin for internalization (11), we hypothesized that G␤␥ association with tubulin might direct these receptors to the clathrin-mediated endocytic pathway. Tubulin binds the essential vesicle-pinching protein, dynamin (for review, see Refs. 31 and 32) and activates dynamin GTPase (33). The mechanoenzyme dynamin is also known to recruit effectors of the endocytic process (34), one of which might be tubulin.
Here we demonstrate that complexes containing m 3 musca-rinic receptors, G␤␥, and tubulin internalize at the offset of PLC␤ 1 signaling through a clathrin-mediated endocytic mechanism. It is suggested that coupling of m 3 receptors to tubulin through G␤␥ might guide receptor sequestration, since tubulin interacts with dynamin and, thus, might facilitate the insertion of the complexes in the clathrin-coated pits. This may represent a novel mechanism for receptor internalization that is alternative to the ␤-arrestin-mediated pathway. It is finally hypothesized that, if excluded from the early endosomes, tubulin-G␤␥ complexes might seed tubulin assembly in the cytosol and reverse the microtubule depolymerization caused by PLC␤ 1evoked Ca 2ϩ increase. This suggests a regulated interconnection between G protein-mediated intracellular signaling and the remodeling of the cytoskeleton.

EXPERIMENTAL PROCEDURES
Expression Constructs-cDNAs encoding HA-tagged wild type and dominant negative K44E dynamin I were originally obtained from Dr. Richard Vallee (Columbia University) (35). They were subsequently cloned into pcDNA3.1zeo using XhoI and XbaI and kindly provided by Dr. Mark von Zastrow (University of California, San Francisco) (36).
Cell Culture and Transfection-SK-N-SH neuroblastoma cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum according to standard procedures (4). Where indicated cells were seeded in 12-well plates and transfected with 1 g/well cDNA of wild type or dominant negative dynamin I K44E using Gene-PORTER transfection reagent (GTS, Inc.) according to the manufacturer's instruction. Assays were performed 48 h after the start of transfection. Protein expression was verified by immunoblotting.
Membrane Preparation and Western Blotting-SK-N-SH cells were sonicated in ice-cold 20 mM Hepes, pH 7.4, 1 mM MgCl 2 , 100 mM NaCl, 1 mM dithiothreitol, 0.3 mM phenylmethylsulfonyl fluoride (3 ϫ 30 s, Branson Sonifer). Membrane pellets and supernatants were prepared as described (37). Protein concentration was measured by the Bradford dye binding assay (38) with bovine serum albumin as a standard. Membrane proteins transferred to polyvinylidene difluoride membranes (0.45 m, Millipore Corp.) were probed with monoclonal antibody specific for ␣-tubulin (DM1A, Sigma) at a dilution of 1:1000 and polyclonal antisera specific for G␣ q , G␤, m 3 muscarinic receptors, clathrin, and HA (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:500. Anti-mouse, rabbit, or goat IgGs were used as secondary antisera, respectively, followed by ECL detection of the corresponding protein bands.
Translocation of Signaling Proteins in Intact SK-N-SH Cells-SK-N-SH cells were collected and washed three times with phosphatebuffered saline, and aliquots of 1 ϫ 10 7 cells were distributed in plastic tubes on ice. Carbachol (100 M) was immediately added, and the samples were incubated for the indicated time periods in a water bath at 37°C with constant shaking. When tested, atropine (10 M) was added before carbachol. After the incubation, each sample was transferred on ice and immediately sonicated as described. The samples were centrifuged at 600 ϫ g at 4°C, and the supernatants were pelleted at 20,000 ϫ g at 4°C. Both the pellets (P2 membrane fractions) and the corresponding supernatants (crude cytosolic fractions) were subjected to SDS/PAGE and immunoblotting. Note that the crude cytosolic fraction contains certain cellular structures including endocytic vesicles. Monoclonal anti-tubulin antibody (DM1A, Sigma), rabbit polyclonal anti-G␣ q and G␤␥, and goat polyclonal anti-clathrin (Santa Cruz) antisera and ECL detection were utilized. The results were analyzed in a Storm 840 (Molecular Dynamics) image analysis system.
Confocal Immunofluorescence Microscopy-SK-N-SH neuroblastoma cells were treated with 100 M carbachol, fixed in Ϫ20°C methanol for 3 min, and washed 3 times 10 min each with phosphate-buffered saline containing 0.1% Triton X-100. The cells were blocked for 40 min in phosphate-buffered saline containing 5% milk and washed in phosphate-buffered saline. The cells were incubated with primary polyclonal anti-G␤, G␣ q , clathrin, m 3 muscarinic receptor, or HA antisera (Santa Cruz) and secondary fluorescein isothiocyanate (FITC)-conjugated, tetramethylrhodamine isothiocyanate (TRITC)-conjugated, or Texas Redconjugated secondary antisera as indicated. When a monoclonal anti-␣-tubulin antibody (DM1A, Sigma) was utilized, secondary Texas Redconjugated anti-mouse antibody was used. All primary polyclonal and secondary antisera were used at a dilution of 1:100. The monoclonal anti-tubulin antibody was utilized at a dilution of 1:500. For each experimental condition a total of 60 randomly selected cells over three to six consecutive experiments were evaluated for the distribution and colocalization of G␣ q , G␤, clathrin and tubulin, or tubulin and m 3 muscarinic receptors or dynamin. Images were acquired using a laserscanning confocal microscope Zeiss LSM 510 equipped with a 63ϫ water immersion objective. A 488-nm beam from an argon-krypton laser was used for the excitation of FITC, whereas a 543-nm beam was used for Texas Red/TRITC excitation. Emission from FITC was detected through a BP505 filter whereas emission was from Texas Red/TRITC through a LP560 filter. Areas of antibody colocalization appear in yellow. Differential interference contrast (DIC) images of the cells were regularly acquired as well. Coverslips were examined at random. Final image composites were created using Adobe Photoshop 6.0. No specific TRITC, Texas Red, or FITC labeling was observed in cells treated with preimmune serum instead of the primary antiserum. Texas Red labeling was not observed when the anti-tubulin antibody was preincubated overnight at 4°C with purified tubulin (1:1 ratio). FITC or TRITC labeling was not detected when the anti-G␤ or anti-G␣ q antisera were preincubated with purified G␤␥ or G␣ q , respectively; both conditions tested at the same antibody dilutions. Similarly, no clathrin, G␤, G␣ q , m 3 muscarinic receptor, or HA labeling was observed in the presence of respective blocking peptides provided by the manufacturer. Finally, G␣ q labeling was not detected in mouse embryonic fibroblasts lacking G␣ q/11 (from M. Simon, Caltech).
Immunoprecipitation-Coimmunoprecipitations of tubulin, G␤␥, G␣ q , m 3 muscarinic receptors, and clathrin were tested in radioimmune precipitation assay buffer-stripped SK-N-SH membranes or crude cytosolic fractions adjusted to 1% sodium deoxycholate and 1% Triton X-100 (1 h, 4°C, and constant shaking) before and after stimulation with 100 M carbachol as indicated. Samples (0.5 mg/ml cytosolic protein or 0.1 mg/ml membrane extract) were precleared with normal rabbit, goat, or mouse IgG (depending on the experimental protocol) and protein A/G PLUS-agarose (Santa Cruz) according to the manufacturer's instruction. Isolated supernatants were incubated for 4 h with 8 l of polyclonal goat anti-clathrin or rabbit anti-G␤ antisera (Santa Cruz) or mouse tubulin antibody (DM1A, Sigma) at 4°C with constant shaking. Immune complexes were precipitated with protein A/G PLUSagarose as instructed by the manufacturer, and each immunoprecipitate was washed 4 times with radioimmune precipitation assay buffer. The pellets were resuspended in 1ϫ sample buffer and subjected to SDS/PAGE followed by immunoblotting and ECL detection of the protein bands. Monoclonal anti-tubulin antibody (DM1A, Sigma) (dilution of 1:1000) and polyclonal anti-G␤, G␣ q , clathrin (dilution of 1:500), and anti-m 3 muscarinic receptor (dilution of 1:200) antisera (Santa Cruz) were used to detect proteins of interest as described. No cross-reactivity was observed between the antisera used.
Materials-Carbachol and atropine sulfate were from Sigma. FITCand TRITC-conjugated goat anti-rabbit antisera were from EY Laboratories. FITC-and TRITC-conjugated rabbit anti-goat antisera were from Zymed Laboratories Inc.. Rhodamine-conjugated goat anti-rabbit antiserum from Roche Applied Science and FITC-conjugated rabbit anti-goat antiserum from Vector Laboratories were also utilized. Texas Red-conjugated goat anti-mouse antiserum was from Jackson Laboratories. All other reagents were of analytical grade.

Internalization of Membrane-associated Tubulin with G␤␥, but Not G␣ q , after Carbachol Stimulation of SK-N-SH Neuro-
blastoma Cells-Dimeric tubulin rapidly associates with the plasma membrane in response to carbachol stimulation (4,37). Cellular imaging demonstrates that 15 min after carbachol addition tubulin colocalizes with G␤␥ in vesicle-like structures in the cytosol (8). To analyze biochemically the sequence of these events, temporal patterns of colocalization of tubulin, G␣ q , and G␤␥ at the membrane and in the cytosol were studied in intact SK-N-SH neuroblastoma cells (Fig. 1). Cells were incubated with carbachol for different time periods, and the content of tubulin, G␣ q , and G␤␥ in membrane and cytosolic fractions was analyzed by immunoblotting.
As previously observed (4), tubulin translocated to the plasma membrane in response to m 3 muscarinic receptor activation (Fig. 1A). Two minutes after carbachol addition membrane-associated tubulin increased by 243% (n ϭ 4) and gradually decreased subsequent to this time point. Ten minutes after carbachol addition, two-thirds of the tubulin that had been recruited to the membrane returned to the cytosol.
G␤ distribution between membrane and cytosol also changed during the course of carbachol stimulation (Fig. 1B). Ten minutes after agonist addition, membrane G␤ decreased by 33% (n ϭ 4) compared with the initial quantity of this protein. This corresponded to a G␤ increase in the cytosol. Neither tubulin nor G␤ translocation occurred when the cells were pretreated with 10 M atropine. In contrast, membrane or cytosolic distribution of G␣ q did not significantly change during the course of carbachol stimulation (Fig. 1C). This observation was in line with previous findings showing a lack of G␣ q/11 translocation to the cytosol after receptor stimulation in neurons or in transiently transfected HEK 293 cells (39,40). Thus, although tubulin was recruited to the plasma membrane to transactivate G␣ q in response to carbachol stimulation (4,37), it translocated back to the cytosol with G␤␥ subunits but not G␣ q .
Confocal microscopy was used to verify these findings. The intracellular localization of tubulin, G␣ q , and G␤␥ was studied during the course of carbachol stimulation of intact SK-N-SH cells (Fig. 2). In unstimulated cells, G␣ q and G␤ colocalized at focal spots of the plasma membrane, presumably where G␣ q /␤␥ heterotrimers were located ( Fig. 2A). However, significant G␣ q /G␤ colocalization was also observed in the perinuclear region of the cells. Two minutes after carbachol application, the areas of G␣ q /G␤ colocalization at the plasma membrane appeared somewhat merged and significantly broader. At the 15-min time point, G␣ q and G␤ colocalized predominantly in the perinuclear region. G␤ was also seen in vesicle-like structures in the cytosol. However, G␣ q was not detected at these intracellular locations.
Next, tubulin colocalization with G␣ q or G␤ was evaluated (Fig, 2, B and C). In unstimulated cells tubulin colocalized with G␣ q in the perinuclear area of the cells as well as along microtubules (Fig. 2B). This was concordant with previous observations demonstrating G␣ i1 , G␣ s , and G␣ o decoration of microtubules in cell-free system and in PC12 cells (6,41). G␤ sparsely colocalized with tubulin in untreated SK-N-SH cells. However, 2 min after carbachol addition, tubulin associated with the plasma membrane and colocalized with both G␣ q and G␤ (Fig,  2, B and C). Fifteen minutes post-carbachol, significant colocalization of tubulin and G␤, but not G␣ q , was observed in vesicles in the cytosol. G␣ q was again seen along microtubules. Magnified images of SK-N-SH cells are shown to underline the differences between tubulin/G␤ and tubulin/G␣ q colocalization during the course of carbachol stimulation (Fig. 2D). These findings confirmed the biochemical observations showing a correlation between tubulin and G␤, but not G␣ q translocation to the cytosol (Fig. 1). It appeared that the pool of cytosolic G␣ q was different from that at the plasma membrane, since membrane G␣ q did not appear to internalize in response to agonist stimulation (Refs. 39 and 40 and herein). Thus, it appeared that tubulin engaged in revolving interactions with G␣ q and G␤␥ during the course of PLC␤ 1 signaling.
Tubulin and G␤ Internalize through Clathrin-mediated Endocytic Mechanism-Both biochemical and cellular imaging approaches were utilized to identify the nature of endocytic vesicles containing tubulin and G␤␥. The G␣ q -coupled m 3 muscarinic receptors of neuroblastoma cells internalize through clathrin-mediated endocytosis (42). We tested whether tubulin and/or G␤␥ utilized this endocytic pathway.
Clathrin presence at the membrane and in the cytosol was investigated during the course of carbachol stimulation of intact SK-N-SH cells (Fig. 3). A striking similarity with the pattern of translocation of tubulin and G␤ was observed. Similarly to tubulin, clathrin association with the membrane increased within 2 min of carbachol addition (220%, n ϭ 4). Moreover, clathrin translocated back to the cytosol after 10 min  A, colocalization of G␣ q and G␤. G␣ q appears in green, and G␤ is in red. Areas of G␣ q /G␤ colocalization appear in yellow. In unstimulated cells G␣ q and G␤ colocalized in the perinuclear region and at highly localized sites of the cell membrane. Their membrane colocalization increased after the addition of agonist (2 min). G␤, but not G␣ q , was seen in vesicles in the cytosol (15 min). B, colocalization of tubulin and G␤. G␤ appears in green, and tubulin is in red. Areas of tubulin-G␤ colocalization are seen in yellow. Carbachol-induced tubulin-G␤ colocalization at the cell membrane was greatest after 2 min. The two proteins colocalized in vesicles in the cytosol at 15 min of agonist stimulation. C, colocalization of tubulin and G␣ q . G␣ q appears in green, and tubulin is in red. Areas of tubulin-G␣ q colocalization are seen in yellow. Tubulin and G␣ q colocalized along microtubules. At 2 min of agonist exposure, depolymerization of microtubules and tubulin-G␣ q colocalization at the cell membrane were observed. D, magnified images of control and carbachol-treated SK-N-SH cells labeled for G␤ and G␣ q . Tubulin colocalization with both proteins at the plasma membrane at 2 min of agonist stimulation is apparent. At 15 min G␤ is at the plasma membrane and colocalized with tubulin in vesicle-like structures in the cytosol, whereas G␣ q is at the membrane and along the microtubules, as seen in the unstimulated cells.
of agonist stimulation, as did tubulin and G␤ (Fig. 1). These results suggested agonist-dependent internalization of tubulin and G␤ with the clathrin-coated vesicles.
Clathrin colocalization with tubulin and G␤ in SK-N-SH cells was studied by confocal microscopy (Fig. 4). G␣ q was used as negative control, since it did not internalize (43). In untreated cells clathrin did not colocalize with tubulin, G␤, or G␣ q . However, 2 min after carbachol addition, clathrin translocated to the plasma membrane and colocalized with G␣ q and G␤ (Fig. 4, B and C) as well as membrane-associated tubulin (Fig. 4A). Although areas of colocalization of clathrin and G␣ q were broad and not well defined, colocalization with tubulin and G␤ was found in pit-like regions of the plasma membrane. Vesicles containing clathrin and G␤ as well as clathrin and tubulin were also observed in proximity to the cellular cortex. Fifteen minutes after carbachol stimulation, both tubulin and G␤ were seen in clathrin-coated vesicles throughout the cytosol (Fig. 4, A and B). G␣ q was not present in these cellular structures (Fig. 4C). All carbachol-mediated processes were inhibited by atropine (10 M). Thus, internalization of membraneassociated tubulin and G␤ appeared to proceed through clathrin-mediated endocytosis.
To test if clathrin, tubulin, and G␤␥ were internalized together, coimmunoprecipitation of clathrin with tubulin, G␤, or G␣ q from the cytosol of SK-N-SH cells was tested before and 15 min after carbachol stimulation (Fig. 5). Tubulin, but not G␤, coimmunoprecipitated with clathrin from the cytosol of untreated cells. Fifteen minutes after agonist addition tubulin/ clathrin coimmunoprecipitation increased by 258% (n ϭ 4). Although G␤ also coimmunoprecipitated with cytosolic clathrin after carbachol stimulation, G␣ q did not either before or after agonist addition. This experiment supported the view that carbachol-evoked involvement of tubulin in G␣ q -mediated signaling was followed by tubulin internalization with G␤␥ through a clathrin-mediated endocytic mechanism.
Complexes of m 3 Muscarinic Receptor, G␤␥, and Tubulin Internalize with the Clathrin-coated Vesicles-G␤␥ subunits might mediate tubulin internalization through their interaction with both tubulin (5, 8) and the m 3 muscarinic receptors (22). Although tubulin does not appear to bind to G␣ q -coupled muscarinic receptors (37), G␤␥ interacts with the third intracellular loop of the m 3 type (22). Thus, G␤␥ might "bridge" tubulin internalization with these activated receptors. Under such a scenario, complexes of tubulin-G␤␥-m 3 muscarinic receptors should coimmunoprecipitate with clathrin-coated vesicles from the cytosol of agonist-stimulated cells.
Cytosolic fractions of SK-N-SH cells were tested for coimmunoprecipitation of m 3 muscarinic receptors with G␤, tubulin, and clathrin before and 15 min after carbachol stimulation (Fig. 6). As seen for the m 1 muscarinic receptor (37), tubulin did not coimmunoprecipitate with the m 3 receptors of unstimulated cells. However, 15 min after carbachol addition an increase in coimmunoprecipitation of m 3 receptors with G␤, clathrin, and tubulin from the cytosol was observed. This observation was consistent with the notion that m 3 muscarinic receptor-G␤␥-tubulin complexes might internalize through clathrin-mediated endocytosis after carbachol stimulation of the SK-N-SH cells.
This hypothesis was tested by confocal microscopy. Because m 3 muscarinic receptors interact with G␤␥ (22) and are shown to internalize with clathrin-coated vesicles in neuroblastoma cells (42), their potential agonist-evoked colocalization with tubulin was investigated (Fig. 7). The m 3 receptors did not colocalize with tubulin in untreated SK-N-SH cells. However, 2 min after carbachol addition they colocalized with membraneassociated tubulin. At 15 min of agonist exposure colocalization of m 3 receptors and tubulin was seen in vesicle-like formations in the cytosol and along microtubules. These results supported the view that the internalization of agonist-activated m 3 muscarinic receptors involves membrane-associated tubulin.
Dominant-negative Dynamin Inhibits Tubulin Translocation during Carbachol Stimulation-Because internalization of m 3 muscarinic receptors depends on dynamin (11) and tubulin interacts with this essential endocytic protein (33) we tested whether a dominant-negative dynamin I construct, K44E, would affect tubulin internalization after agonist exposure. Dynamin K44E binds GTP poorly (35) and, thus, does not pinch vesicles from the plasma membrane and inhibits agonistevoked internalization. As seen in Fig. 8, wild type dynamin I colocalized with tubulin at the plasma membrane at 2 min of carbachol stimulation, and both proteins translocated to vesicle-like structures in the cytosol after 15 min of agonist exposure. To the contrary, tubulin did not colocalize with dynamin K44E at the plasma membrane or in vesicles in the cytosol during the course of carbachol stimulation. Thus, similarly to the activated m 3 muscarinic receptors (11), the agonist-evoked translocation of tubulin was a dynamin-dependent process. DISCUSSION Internalization of receptors and other cell surface components in neuronal cells occurs via clathrin-mediated endocytosis, although other less characterized pathways are also reportedly involved. Receptors internalized in clathrin-coated vesicles are subsequently delivered to early endosomes, where they are sorted to recycle back to the plasma membrane or be transported to late endosomes/lysosomes for degradation. Other proteins involved in signal transduction, like GRKs and ␤-arrestins, are also found in the clathrin-coated vesicles (9,44). GRKs phosphorylate activated receptors, which allows for the binding of ␤-arrestins. The latter interact with both the heavy chain of clathrin and the AP2 complex and, thus, sequester activated receptors to the clathrin-coated pits for internalization (9,10).
Similarly to GRKs and ␤-arrestins, tubulin associates with the plasma membrane in response to agonist stimulation (4,37). However, membrane-associated tubulin performs a differ-  Carbachol-induced clathrin-G␤ colocalization was greatest in vesicles in the cytosol 15 min after agonist exposure of the cells. C, lack of colocalization of clathrin and G␣ q . G␣ q appears in green, and clathrin is in red. Areas of clathrin-G␣ q colocalization are seen in yellow. After carbachol stimulation G␣ q was observed at the cell membrane but not in clathrin vesicles in the cytosol.
clarified, a decrease in tubulin at the plasma membrane beginning 5 min after m 1 or m 3 muscarinic receptor stimulation has been observed (4, 37).
Here we demonstrate that tubulin associated with the plasma membrane in response to agonist stimulation of SK-N-SH neuroblastoma cells internalized through clathrin-mediated endocytic mechanism. It also appeared that this tubulin participated in the clathrin-mediated internalization of the m 3 muscarinic receptors, perhaps through their mutual interaction with G␤␥. G␤␥ subunits are multifunctional complexes that are involved in variety of signal transduction mechanisms. Although G␤␥s assist agonist-evoked membrane translocation of GRKs (26), they do not support the membrane association of tubulin in response to agonist stimulation (8). To the contrary G␤␥ appears involved in dissociation of tubulin-GDP from the plasma membrane at the offset of PLC␤ 1 signaling. This hypothesis was tested in the present study.
Both biochemical experiments and cellular imaging indicated plasma membrane colocalization and subsequent cointernalization of tubulin and G␤␥ after carbachol stimulation of SK-N-SH neuroblastoma cells (Fig. 1). The patterns of tubulin and G␤␥ translocation to the cytosol were identical to that of clathrin ( Figs. 1 and 3), and both proteins were found in the clathrin-coated vesicles (Fig. 4). Thus, it appeared that tubulin-G␤␥ complexes might internalize through a clathrin-mediated endocytic mechanism. This was supported by the observation that coimmunoprecipitation of tubulin, G␤, and clathrin from the cytosol of carbachol-stimulated cells increased after 15 min of agonist exposure (Fig. 5). This endocytic mechanism did not involve G␣ q , which did not appear to dissociate from the plasma membrane (Refs. 39 and 40; Fig. 1) and was not found in the clathrin-coated vesicles (Fig. 4). G␣ q -clathrin coimmunoprecipitation from the cytosol was also not observed (Fig. 5).
These findings are supported by previous observations showing association of tubulin and clathrin with the plasma membrane in response to receptor activation (37,45). G␤␥ involvement in clathrin-mediated endocytosis has also been reported since G␤␥ capture by overexpressed G␣ strongly inhibits this process (46). Thus, G␤␥ binding of tubulin at the membrane (8) might engage tubulin in the clathrin-mediated endocytic pathway at the offset of PLC␤ 1 signaling.
Activated m 1 and m 3 receptors sequester through a clathrinmediated endocytocytic mechanism (42), and muscarinic receptors are present in coated vesicles from bovine brain (47)(48)(49). Amino acids 286 -292 in the 3rd intracellular loop of m 1 muscarinic receptors are involved in receptor internalization (50). A similar region in the closely related m 3 receptor (residues 289 -330) binds G␤␥ (22). M 3 receptor constructs lacking G␤␥ binding motifs sequester significantly less than wild type receptors, although they retain ligand recognition properties and the ability to increase intracellular calcium (22). In addition, although agonist-induced phosphorylation and uncoupling of m 3 muscarinic receptors from G␣ q/11 in SH-SY5Y neuroblastoma cells is significantly attenuated by expression of kinase-dead mutant of GRK6, their internalization is not (51). Thus, it appeared that in SK-N-SH cells, G␤␥ subunits rather than GRK/␤-arrestin might be involved in internalization of m 3 receptors with clathrin-coated vesicles. It is noteworthy in this regard that, although G q -coupled muscarinic receptors interact with G␤␥ (22), they do not appear to interact directly with tubulin (37). G␤␥ could bridge these molecules.
The possible link between internalization of G␤␥-tubulin complexes and m 3 muscarinic receptors was investigated. Although tubulin did not colocalize with m 3 muscarinic receptors of unstimulated cells, both were found in vesicles in the cytosol 15 min after carbachol stimulation (Fig. 7). Moreover, m 3 receptors coimmunoprecipitated with G␤␥, tubulin, and clathrin from cytosol of agonist-stimulated cells (Fig. 6). This suggested that m 3 muscarinic receptor-G␤␥-tubulin complexes internalized through the clathrin-mediated endocytic pathway. These observations supported the view that G␤␥ subunits might serve as docking units within larger protein assemblies involved in sequestration of m 3 muscarinic receptors (22).
Internalization of m 1 , m 3 , and m 4 muscarinic as well as 5-hydroxytriptamine 2A (5-HT 2A ) or gonadotropin-releasing hormone (GnRH) receptors is reported to depend on dynamin but not ␤-arrestin (11,13,52). The third intracellular loop of m 3 receptors appears responsible for this arrestin-resistant, dynamin-sensitive internalization (11). Because G␤␥ binds to this region of m 3 receptors (22), it might assist receptor sequestration in the clathrin-coated pits instead of ␤-arrestin. Both G␤␥ subunits and ␤-arrestins have scaffolding properties (22,53). We propose that tubulin is an indispensable part of m 3 muscarinic receptor sequestration since it binds both G␤␥ (5,8) and dynamin (33). The finding that dominant-negative dynamin inhibits tubulin translocation in response to agonist stimulation (Fig. 8) supports this hypothesis. Thus, tubulin-G␤␥ complexes might recruit m 3 muscarinic receptors to the clathrincoated pit assembly instead of ␤-arrestin. It should be noted, however, that ␤-arrestinand dynamin-dependent internalization of m 2 -m 5 muscarinic receptors has also been reported (54). Cell type-or receptor type-specific differences in intracellular signaling mechanisms might account for this discrepancy. It is also possible that both mechanisms operate in the cell depending on the stimulus or specific experimental conditions.
It is also noteworthy that phosphoinositide-binding proteins like ␤-arrestin tend to concentrate in clathrin-coated vesicles (55). Tubulin binds phosphatidylinositol 4,5-bisphosphate, and this interaction is instrumental in tubulin association with the plasma membrane and regulation of PLC␤ 1 signaling (7). Such interaction might also contribute to the m 3 receptor-G␤␥-tubulin sequestration in the clathrin-coated pits of the plasma membrane. Phosphatidylinositol 4,5-bisphosphate dependence of agonist-evoked endocytosis of m 3 receptors in SH-SY5Y neuroblastoma cells has been demonstrated (56).
In conclusion this study presents evidence that tubulin involved in the regulation of PLC␤ 1 signaling after agonist stimulation of SK-N-SH neuroblastoma cells internalized through a clathrin-mediated endocytic mechanism. This tubulin was apparently involved in the ␤-arrestin-independent, but dynamindependent internalization of m 3 muscarinic receptors through its interaction with both G␤␥ and dynamin. In turn, G␤␥ interaction with both m 3 muscarinic receptors and tubulin might scaffold internalizing complexes. G␣ q did not appear to undergo clathrin-mediated internalization, although it is transactivated by tubulin after agonist stimulation. It is proposed that G␤␥ and tubulin are instrumental in ␤-arrestin-independent mechanisms of receptor internalization that involve dynamin. This novel regulatory mechanism might add to the understanding of receptor internalization as well as cross-regulation between intracellular signaling and the dynamics of the microtubule cytoskeleton.