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• W2049587780 abstract "Although agonist-dependent endocytosis of G protein-coupled receptors (GPCRs) as a means to modulate receptor signaling has been widely studied, the constitutive endocytosis of GPCRs has received little attention. Here we show that two prototypical class I GPCRs, the β2 adrenergic and M3 muscarinic receptors, enter cells constitutively by clathrin-independent endocytosis and colocalize with markers of this endosomal pathway on recycling tubular endosomes, indicating that these receptors can subsequently recycle back to the plasma membrane (PM). This constitutive endocytosis of these receptors was not blocked by antagonists, indicating that receptor signaling was not required. Interestingly, the G proteins that these receptors couple to, Gαs and Gαq, localized together with their receptors at the plasma membrane and on tubular recycling endosomes. Upon agonist stimulation, Gαs and Gαq remained associated with the PM and these endosomal membranes, whereas β2 and M3 receptors now entered cells via clathrin-dependent endocytosis. Deletion of the third intracellular loop (i3 loop), which is thought to play a role in agonist-dependent endocytosis of the M3 receptor, had no effect on the constitutive internalization of the receptor. Surprisingly, with agonist, the mutated M3 receptor still internalized and accumulated in cells but through clathrin-independent and not clathrin-dependent endocytosis. These findings demonstrate that GPCRs are versatile PM proteins that can utilize different mechanisms of internalization depending upon ligand activation. Although agonist-dependent endocytosis of G protein-coupled receptors (GPCRs) as a means to modulate receptor signaling has been widely studied, the constitutive endocytosis of GPCRs has received little attention. Here we show that two prototypical class I GPCRs, the β2 adrenergic and M3 muscarinic receptors, enter cells constitutively by clathrin-independent endocytosis and colocalize with markers of this endosomal pathway on recycling tubular endosomes, indicating that these receptors can subsequently recycle back to the plasma membrane (PM). This constitutive endocytosis of these receptors was not blocked by antagonists, indicating that receptor signaling was not required. Interestingly, the G proteins that these receptors couple to, Gαs and Gαq, localized together with their receptors at the plasma membrane and on tubular recycling endosomes. Upon agonist stimulation, Gαs and Gαq remained associated with the PM and these endosomal membranes, whereas β2 and M3 receptors now entered cells via clathrin-dependent endocytosis. Deletion of the third intracellular loop (i3 loop), which is thought to play a role in agonist-dependent endocytosis of the M3 receptor, had no effect on the constitutive internalization of the receptor. Surprisingly, with agonist, the mutated M3 receptor still internalized and accumulated in cells but through clathrin-independent and not clathrin-dependent endocytosis. These findings demonstrate that GPCRs are versatile PM proteins that can utilize different mechanisms of internalization depending upon ligand activation. G protein-coupled receptors (GPCRs) 2The abbreviations used are: GPCR, G protein-coupled receptor; β2R, β2 adrenergic receptor; Carb, carbachol; CDE, clathrin-dependent endocytosis; CIE, clathrin-independent endocytosis; Gαq, α-subunit of the heterotrimeric Gq protein; Gαs, α-subunit of the heterotrimeric Gs protein; G protein, heterotrimeric guanine nucleotide-binding protein; HA, hemagglutinin; [3H]NMS, N-[3H]methylscopolamine; i3 loop, the third intracellular loop of G protein-coupled receptors; Iso, isoproterenol; MHCI, major histocompatibility complex class I protein; M3R, M3 muscarinic receptor; M3-short, M3R where most of the i3 loop was deleted, from Ala-303 to Thr-499; PM, plasma membrane; Tf, transferrin; TfR, transferrin receptor; PBS, phosphate-buffered saline; GFP, green fluorescent protein. belong to a superfamily of seven transmembrane-spanning proteins that respond to a diverse array of sensory and chemical stimuli (1Bockaert J. Pin J.P. EMBO J.. 1999; 18: 1723-1729Google Scholar, 2Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell Biol.. 2002; 3: 639-650Google Scholar, 3Kristiansen K. Pharmacol. Ther.. 2004; 103: 21-80Google Scholar, 4Foord S.M. Bonner T.I. Neubig R.R. Rosser E.M. Pin J.P. Davenport A.P. Spedding M. Harmar A.J. Pharmacol. Rev.. 2005; 57: 279-288Google Scholar). Activation of GPCRs through the binding of specific agonists induces conformational changes that allow activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) (5Oldham W.M. Hamm H.E. Nat. Rev. Mol. Cell Biol.. 2008; 9: 60-71Google Scholar, 6Deupi X. Kobilka B. Adv. Protein Chem.. 2007; 74: 137-166Google Scholar). To ensure that the signals are controlled in magnitude and duration, activated GPCRs are rapidly desensitized through phosphorylation carried out by G protein-coupled receptor kinases (GRKs) (7Tobin A.B. Butcher A.J. Kong K.C. Trends Pharmacol. Sci.. 2008; 29: 413-420Google Scholar). This facilitates β-arrestin binding and promotes receptor uncoupling from the G protein (8Reiter E. Lefkowitz R.J. Trends Endocrinol. Metab.. 2006; 17: 159-165Google Scholar, 9Moore C.A. Milano S.K. Benovic J.L. Annu. Rev. Physiol.. 2007; 69: 451-482Google Scholar). In addition to its role in GPCRs desensitization, β-arrestins promote the translocation of the receptor to the endocytic machinery involving clathrin and adaptor protein-2 (AP-2), thereby facilitating receptor removal from the plasma membrane (10Tsao P. Cao T. von Zastrow M. Trends Pharmacol. Sci.. 2001; 22: 91-96Google Scholar, 11Ferguson S.S. Pharmacol. Rev.. 2001; 53: 1-24Google Scholar, 12Hanyaloglu A.C. von Zastrow M. Annu. Rev. Pharmacol. Toxicol.. 2008; 48: 537-568Google Scholar, 13Marchese A. Paing M.M. Temple B.R. Trejo J. Annu. Rev. Pharmacol. Toxicol.. 2008; 48: 601-629Google Scholar, 14Hamdan F.F. Rochdi M.D. Breton B. Fessart D. Michaud D.E. Charest P.G. Laporte S.A. Bouvier M. J. Biol. Chem.. 2007; 282: 29089-29100Google Scholar, 15Mundell S.J. Luo J. Benovic J.L. Conley P.B. Poole A.W. Traffic.. 2006; 7: 1420-1431Google Scholar). Once internalized, some GPCRs may even continue to signal from endosomes (16Shenoy S.K. Drake M.T. Nelson C.D. Houtz D.A. Xiao K. Madabushi S. Reiter E. Premont R.T. Lichtarge O. Lefkowitz R.J. J. Biol. Chem.. 2006; 281: 1261-1273Google Scholar). Although GPCR internalization is generally considered to be an agonist-dependent phenomenon, some evidence suggests that GPCRs can be endocytosed even in the absence of agonist, a process known as constitutive internalization (17McDonald N.A. Henstridge C.M. Connolly C.N. Irving A.J. Mol. Pharmacol.. 2007; 71: 976-984Google Scholar, 18Mohammad S. Baldini G. Granell S. Narducci P. Martelli A.M. Baldini G. J. Biol. Chem.. 2007; 282: 4963-4974Google Scholar, 19Wolfe B.L. Marchese A. Trejo J. J. Cell Biol.. 2007; 177: 905-916Google Scholar, 20Stanasila L. Abuin L. Dey J. Cotecchia S. Mol. Pharmacol.. 2008; 74: 562-573Google Scholar). The role of constitutive internalization of GPCRs is not clear. One interesting study on cannabinoid CB1 receptors in neurons has shown that constitutive internalization from the somatodendritic and not axonal membrane is responsible for the overall redistribution of receptors from the somatodentritic to the axonal membrane (17McDonald N.A. Henstridge C.M. Connolly C.N. Irving A.J. Mol. Pharmacol.. 2007; 71: 976-984Google Scholar). Another study on the melanocortin MC4 receptor raised the possibility that constitutive endocytosis could be a consequence of the basal activity of the receptor (18Mohammad S. Baldini G. Granell S. Narducci P. Martelli A.M. Baldini G. J. Biol. Chem.. 2007; 282: 4963-4974Google Scholar). Even less is known about the potential trafficking of the transducer of GPCR signaling, the G protein (21Marrari Y. Crouthamel M. Irannejad R. Wedegaertner P.B. Biochemistry.. 2007; 46: 7665-7677Google Scholar). Generally, the binding of the agonist to the GPCR promotes the exchange of GDP on the Gα protein for GTP and allows the dissociation of the trimeric G protein into Gα-GTP and Gβγ dimer subunits (5Oldham W.M. Hamm H.E. Nat. Rev. Mol. Cell Biol.. 2008; 9: 60-71Google Scholar, 22Wess J. Pharmacol. Ther.. 1998; 80: 231-264Google Scholar). Then, the activated G proteins target different effectors (23Cabrera-Vera T.M. Vanhauwe J. Thomas T.O. Medkova M. Preininger A. Mazzoni M.R. Hamm H.E. Endocr. Rev.. 2003; 24: 765-781Google Scholar, 24Johnston C.A. Siderovski D.P. Mol. Pharmacol.. 2007; 72: 219-230Google Scholar). G proteins are localized primarily to the PM where they interact with GPCRs; however, it is not known whether G proteins always remain at the PM or whether they might move into cells along endocytic pathways. Previous work showed that Gαs does not colocalize with β2 receptor on internal compartments after agonist stimulation, but the cellular distribution of Gαs was not examined (25Allen J.A. Yu J.Z. Donati R.J. Rasenick M.M. Mol. Pharmacol.. 2005; 67: 1493-1504Google Scholar). In general, cargo proteins at the plasma membrane (PM) enter the cell through a variety of endocytic mechanisms that can be divided into two main groups: clathrin-dependent endocytosis (CDE) and clathrin-independent endocytosis (CIE). CDE is used by PM proteins such as the transferrin receptor (TfR) that contain specific cytoplasmic sequences recognized by adaptor proteins allowing a rapid and efficient internalization through clathrin-coated vesicles (26Conner S.D. Schmid S.L. Nature.. 2003; 422: 37-44Google Scholar, 27Le Roy C. Wrana J.L. Nat. Rev. Mol. Cell Biol.. 2005; 6: 112-126Google Scholar). In contrast, CIE is used by PM proteins that lack adaptor protein binding sequences including cargo proteins such as the major histocompatibility complex class I protein (MHCI), the glycosylphosphatidylinositol-anchored protein CD59, and integrins (28Brown F.D. Rozelle A.L. Yin H.L. Balla T. Donaldson J.G. J. Cell Biol.. 2001; 154: 1007-1017Google Scholar, 29Mayor S. Pagano R.E. Nat. Rev. Mol. Cell Biol.. 2007; 8: 603-612Google Scholar, 30Naslavsky N. Weigert R. Donaldson J.G. Mol. Biol. Cell.. 2004; 15: 3542-3552Google Scholar). In HeLa cells CIE is independent of, and CDE dependent on, clathrin and dynamin and thus the two different endocytic pathways are distinct and well defined (31Donaldson J.G. Porat-Shliom N. Cohen L.A. Cell Signal.. 2009; 21: 1-6Google Scholar). After internalization in separate vesicles, MHCI-containing vesicles from CIE and transferrin receptor-containing vesicles from CDE subsequently fuse with the early endosomal compartment that is associated with Rab5 and the early endosomal antigen 1 (EEA1) (32Naslavsky N. Weigert R. Donaldson J.G. Mol. Biol. Cell.. 2003; 14: 417-431Google Scholar). TfR is recycled back out to the PM in Rab4- and Rab11-dependent processes. In contrast, some MHCI is trafficked on to late endosomes and lysosomes for degradation, and some is recycled back out to the PM along tubular endosomes that lack TfR and emanate from the juxtanuclear area. Recycling of MHCI back to the PM requires the activity of Arf6, Rab22, and Rab11 (33Radhakrishna H. Donaldson J.G. J. Cell Biol.. 1997; 139: 49-61Google Scholar, 34Weigert R. Yeung A.C. Li J. Donaldson J.G. Mol. Biol. Cell.. 2004; 15: 3758-3770Google Scholar). In this study, we analyzed the trafficking of GPCRs and their G proteins in the presence and absence of agonist in HeLa cells. We examined the trafficking of two prototypical class I GPCRs: the β2 adrenergic receptor (coupled to Gαs) and the M3 acetylcholine muscarinic receptor (coupled to Gαq). We find that β2 and M3 receptors traffic constitutively via CIE, and then, in the presence of agonist, they switch to the CDE pathway. We also examined the role of the third intracellular loop of the M3 receptor in this process. To our knowledge, this study represents the most comprehensive analysis of constitutive trafficking of class I GPCRs and related Gα proteins. We demonstrate that GPCRs are versatile PM cargos that utilize different mechanisms of internalization depending upon ligand activation. Considering the high level of homology between class I GPCRs, this evidence could be applicable to the other members of this family. Materials and Antibodies—Carbamylcholine chloride (carbachol), isoproterenol hydrochloride, atropine sulfate, and propranolol hydrochloride were obtained from Sigma. N-[3H]Methylscopolamine ([3H]NMS, 79–83 Ci/mmol) was from PerkinElmer Life Sciences (Waltham, MA). The mouse monoclonal anti-HA antibody 16b12 (IgG1) was from Covance (Berkeley, CA) and a rabbit anti-HA antibody from Abgent (San Diego, CA). Mouse monoclonal antibody to human MHCI (W6/32) (IgG2a) (Naslavsky et al., Ref. 32Naslavsky N. Weigert R. Donaldson J.G. Mol. Biol. Cell.. 2003; 14: 417-431Google Scholar) was described previously. The mouse anti-clathrin heavy chain was purchased from BD Biosciences (Palo Alto, CA). A mouse anti-Lamp1 antibody (H4A3) was from Developmental Studies Hybridoma Bank (Iowa City, IA). The antibodies against the endogenous Gαs and Gαq subunits were kindly provided by Dr. A. Spiegel (Albert Einstein College of Medicine, Bronx, NY) and were previously described (54Goldsmith P. Gierschik P. Milligan G. Unson C.G. Vinitsky R. Malech H.L. Spiegel A.M. J. Biol. Chem.. 1987; 262: 14683-14688Google Scholar). Invitrogen (Carlsbad, CA) was the source for transferrin (Tfn) conjugated to Alexa-594 and Alexa-conjugated (488, 594, and 680) fluorescent secondary goat-anti-rabbit, goat-anti-mouse (GAM), and isotype-specific GAM-IgG1 and GAM-IgG2a antibodies. Cell Culture, DNA Constructs, and siRNA—HeLa and COS-7 cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin, and 100 units/ml penicillin at 37 °C with 5% CO2. For transfection, cells were plated and transfected the next day by using FuGENE (Roche Applied Science, Indianapolis, IN) following the manufacturer's instructions. Experiments were performed 18–20 h after transfection. The 3HA-tagged receptor constructs, hβ2, hM3, and hM2 receptors (in the plasmid vector pcDNA 3.1 +) were purchased from UMR cDNA Resource Center (Rolla, MO). The construct β2-GFP used in live cell imaging experiments was kindly provided by Dr. J. Benovic (University of Pennsylvania, Philadelphia, PA) and was previously described (35Kallal L. Gagnon A.W. Penn R.B. Benovic J.L. J. Biol. Chem.. 1998; 273: 322-328Google Scholar). The 3HA-tagged hM3-short (where most of the i3 loop of M3 was deleted, from Ala-303 to Thr-499) plasmid was kindly provided by Dr. J. Wess (NIDDK, National Institutes of Health, Bethesda, MD) and it was previously described (36Maggio R. Barbier P. Fornai F. Corsini G.U. J. Biol. Chem.. 1996; 271: 31055-31060Google Scholar). To knockdown clathrin, we used the SMART pool siRNA (a mixture of 4 different siRNA) from Dharmacon. In particular, the four target sequences designed to knock down clathrin were GAGAAUGGCUGUACGUAAU, UGAGAAAUGUAAUGCGAAU, GCAGAAGAAUCAACGUUAU, and CGUAAGAAGGCUCGAGAGU. The final concentration of the pool siRNA in our experiment was 75 nm (18.7 nm for each single siRNA). For the siRNA clathrin knockdown experiments, we followed the double hit siRNA procedure of Motley et al. (55Motley A. Bright N.A. Seaman M.N. Robinson M.S. J. Cell. Biol.. 2003; 162: 909-918Google Scholar). In brief, we seeded HeLa cells at a density of 500,000 cells per 10-cm dish and after 6 h the first siRNA transfection was performed, using Oligofectamine (Invitrogen) and OPTI-MEM I (Invitrogen). Then, on Day 2, a second siRNA transfection was performed. On day 2, 6–8 h before the second treatment of siRNA, we transfected our constructs hβ2, hM3, and hM3-short receptors following our standard procedure with FuGENE. The cells were trypsinized on Day 3 and split in 2 dishes (one for immunofluorescence and one the Western blotting). On day 4, the experiment was performed. Immunofluorescence, Antibody Internalization, and Live Cell Imaging—For immunofluorescence staining, cells were plated on to glass coverslips and transfected the following day. Eighteen hours after transfection, cells were preincubated at 4 °C for 1 h with the mouse anti-HA antibody (IgG1) to label the receptor on the plasma membrane. After washing, cells were incubated at 37 °C at different times in the presence of the mouse anti-MHCI antibody (IgG2a) or in the presence of transferrin (Alexa 594-transferrin), with or without the agonist, to allow internalization. For M3 and M2 muscarinic receptors Carbachol (1 mm) was used, while for β2 receptor isoprotenerol (1 mm) was used as agonists. The cells were fixed in 2% formaldehyde in PBS for 10 min, washed with PBS containing 10% FBS (PBS/FBS) and then incubated at room temperature for 1 h with ∼0.08 mg/ml of unlabeled goat anti-mouse (GAM) in the absence of saponin to block surface antibodies from secondary reagents. After washing, fluorescently-conjugated isotype-specific secondary antibodies (488 GAM-IgG1 and 594 GAM-IgG2a) were used in the presence of 0.2% saponin to detect the internalized receptor and MHCI, respectively. When 594-transferrin was present, we utilized only the secondary antibodies 488 GAM (IgG1). All images were obtained using a 510 LSM confocal microscope (Carl Zeiss, Thornwood, NY) with 63× 1.3 numerical aperture PlanApo objective. Unless indicated, the optical section was less than 1 μm. After acquisition, images were handled using Adobe Photoshop (Adobe Systems, San Jose, CA). All experiments were confirmed at least three times, and a representative image is shown. For live cell imaging, HeLa cells were plated onto Lab-Tek coverglass chambers (Nalge Nunc International, Rochester, NY) and transfected with β2-GFP constructs. Eighteen hours after transfection, cells were imaged on a 37 °C stage in CO2-independent media. Images were acquired every 10 s for 15 min. After 3–5 min, Alexa 594-transferrin was added to the medium with or without isoprotenerol (1 mm). Quantification of the Internalized Receptor with the Single Cell-based Method—To determine and quantify the amount of internal cargo at different times of internalization, we used a single cell-based method. This method allows us to measure the percentage of the internal cargo compared with the total (surface and internalized) for each cell examined. After preincubation at 4 °C for 1 h with the mouse anti-HA antibody (IgG1) to label the receptor (M3 or β2) on the plasma membrane, cells were incubated at 37 °C at different times in the presence or absence of ligand. Then, the cells were fixed, washed, and incubated at room temperature for 1 h with isotype-specific secondary antibody 488 GAM-IgG1 without saponin just to label the receptor left on the PM after the internalization experiment. Next, we added ∼0.08 mg/ml of the unlabeled GAM blocking solution without saponin for 1 h to quench any remaining sites on the surface-bound mouse antibodies. Finally, we used the secondary antibody 594 GAM-IgG1 for 1 h in the presence of saponin to label the internalized receptor. In parallel to each quantification experiment, we performed a separate experiment to set up the acquisition parameters of the images so that the fluorescent signals of the two different secondary antibodies 594 GAM-IgG1 and 488 GAM-IgGI were similar. To do this, we used the two secondary antibodies, 594 GAM-IgG1 and 488 GAM-IgG1, together at the same concentration to label the primary antibody bound to the receptor in a control experiment. After this preliminary set up, all the images were taken with identical acquisition parameters and the fluorescent signals for each measurement was within the dynamic range. For these experiments, the 40× plan Apo objective was used with the pinhole completely open (optical section was about 12 μm) during image acquisition. For each treatment, we quantified the fluorescence of 50–100 cells using Metamorph 4.6. This method allowed us to measure the percentage of the internal cargo compared with the total (surface and internalized). Importantly, when we switched the secondary antibodies and used 594 GAM-IgG1 first to stain the surface receptor and then 488 GAM-IgG1 to label the internal receptor, the results obtained were similar (data not shown). In this analysis, cells expressing high levels of receptor (5-fold over that exhibited by an average cell) that gave a fluorescent signal above the dynamic range were not included (about 20–25% of the cells). Importantly, there was minimal overlap of “surface” and “internally” detected antibody when the cells were viewed by a thin confocal slice. Quantification of the Internalized Receptor with Radioligand Binding Studies—In the radioligand binding assays, 24 h after transfection with M3 receptor, the cells were split into 6-well plates. The next day, the cells were incubated at 37 °C with or without carbachol (1 mm) at different times (from 5 min to 60 min). After drug treatment, the cells were cooled on ice and washed three times with PBS (10-min incubation for each washing step). The loss of cell surface M3 receptor in the presence of carbachol compared with the control (without ligand) was detected by incubating the cells with the cell-impermeant muscarinic ligand [3H]NMS (2 nm) for 2 h at 4 °C. The cells were then washed three times (10-min incubation for each washing step) with ice-cold PBS and solubilized with 1% Triton X-100 in PBS for 10 min. Then, the cells were scraped, and the extracts were transferred to a vial with scintillation fluid, and the radioactivity was measured. Nonspecific binding was assessed as binding remaining in the presence of 10 mm atropine and was subtracted from all the samples. Receptor internalization was defined as the loss of binding of the cell-impermeant [3H]NMS after carbachol treatment compared with nontreated cells. Each experiment was done in triplicate and the experiment was repeated two additional times. Binding data were analyzed using the nonlinear curve-fitting program Prism 4.0b (GraphPad). GPCRs Display Constitutive and Agonist-dependent Internalization through CIE and CDE, Respectively—To study both constitutive and ligand-dependent GPCR endocytosis, we expressed and examined two GPCRs in HeLa cells, the β2-adrenergic receptor, and the M3 muscarinic receptor, which couple to Gs and Gq, respectively. These receptors were tagged at their extracellular, N termini with a triple HA tag allowing us to follow the internalization of the receptor into cells. Studies of GPCR trafficking and function typically employ expression of epitope-tagged receptors in a heterologous system due to the complexity of overlapping receptor subtypes in native systems (37Nathanson N.M. Pharmacol. Ther.. 2008; 119: 33-43Google Scholar). To determine and quantify the amount of internal cargo at different times of internalization, we used a single cell-based method. This method allows us to measure the percentage of the internalized receptor compared with the total receptor (surface and internalized) for each cell examined (we measured about 50–100 cells for each treatment; see “Experimental Procedures” for details). When we examined the trafficking of the β2 receptor using antibody internalization, β2 was internalized into cells in the absence of agonist (Fig. 1A). This constitutive internalization was apparent at 5 min and increased further at 30 min. Quantification of the internalized receptor revealed that about 10% of the initially bound material was inside the cell at 5 min and 20% at 30 min (Fig. 1C). The addition of agonist (1 mm Iso) increased the amount of β2 receptor internalized and this could be observed in fluorescence images (Fig. 1B). About 36% of surface β2 receptor was internalized in 5 min and 75% at 30 min (Fig. 1, B and C). Similar observations were made with the M3 receptor by imaging (not shown) and quantification of antibody internalization (see below). The amount of the GPCR internalized with ligand measured with the single cell-based method was more compared with the amount obtained using biochemical methods (supplemental Fig. S1 and see “Experimental Procedures” for details) or reported by other groups (38Hanyaloglu A.C. von Zastrow M. J. Biol. Chem.. 2007; 282: 3095-3104Google Scholar). One explanation for this is that biochemical assays measure total receptor internalization of all cells whereas our method of quantification excludes cells expressing very high levels of receptor. Thus biochemical methods could under estimate receptor internalization compared with the cell-based method we employed. To investigate if the constitutive internalization of the β2 and M3 receptors was a consequence of their basal activity or the presence of low concentration of ligand in the culture medium, we used the antagonists (inverse agonists) propranolol and atropine, respectively, to block this activity. Neither propranolol nor atropine affected the constitutive receptor internalization of β2 or M3 at 30 min (supplemental Fig. S2). To determine the pathway of constitutive internalization of β2 and M3 receptors, we compared the endocytosis of these receptors with two endogenous PM proteins, MHCI and TfR, that are internalized by CIE and CDE, respectively, using antibodies to MHCI and Alexa594-conjugated transferrin (Tf). At 5 min of internalization in the absence of agonist, β2 (Fig. 2A) and M3 (supplemental Fig. S3A) colocalized with MHCI but not with Tf on peripheral endosomal structures and on recycling tubular endosomes. The presence of M3 and β2 receptors on recycling tubular endosomes that colocalized with MHC I was especially pronounced in cells treated with inhibitors of actin polymerization (data not shown); such treatments block recycling of CIE cargo back to the PM (33Radhakrishna H. Donaldson J.G. J. Cell Biol.. 1997; 139: 49-61Google Scholar, 34Weigert R. Yeung A.C. Li J. Donaldson J.G. Mol. Biol. Cell.. 2004; 15: 3758-3770Google Scholar). By contrast, in the presence of agonist for 5 min, internalized β2 (Fig. 2B) and M3 (supplemental Fig. S3B) receptors colocalized with Tf and not with MHCI. Even at longer times with agonist (30 min), β2 and M3 did not localize to the recycling tubular endosomes (data not shown). We also found that constitutive internalization of β2 and M3 receptors was not inhibited by the inhibitory mutant of dynamin 2, K44A, whereas ligand-activated internalization was impaired (data not shown). Taken together, these observations suggest that in the absence of ligand, both β2 and M3 internalized and colocalized with proteins that enter cells via a CIE pathway and then upon agonist stimulation, these receptors were found in compartments containing CDE cargo. Having observed a shift in endocytic pathways used by these receptors upon addition of ligand in fixed cells, we wished to examine this in living cells. β2R-GFP expressed in HeLa cells mostly localized to the PM, on internal vesicles and on endosomal recycling compartments of the CIE pathway (supplemental Fig. S4). To study the mechanism of internalization of this receptor, we imaged the internalization of Tf-594 in cells expressing a GFP-tagged version of the β2 receptor. Without agonist, β2R-GFP and Tf were not observed together during the 15-min incubation (Fig. 2C, and supplemental Movie S1). By contrast, in the presence of ligand, β2R-GFP, and endocytosed Tf were present together in the same endosome (Fig. 2D, and supplemental Movie S2). These results demonstrate that β2 and M3 traffic constitutively in the CIE pathway, and then, in the presence of agonist switch to the CDE pathway. Similar results were obtained in COS-7 cells (data not shown). To confirm the differences between the constitutive and agonist-dependent GPCR endocytosis, we depleted clathrin from HeLa cells using siRNA. As shown by immunoblot and by immunofluorescence, clathrin was 85–90% depleted (supplemental Fig. S5) and we observed that endocytosis of Tf was blocked in cells depleted of clathrin (data not shown). Under these conditions, the constitutive internalization of β2 receptors was slightly inhibited by the depletion of clathrin (Fig. 3, A and C). By contrast, the agonist-dependent endocytosis of β2 was strongly affected by the knock-down of clathrin (Fig. 3, B and C). Endocytosis in the presence of the agonist was reduced in cells depleted of clathrin to the level observed for endocytosis in the absence of ligand. Similar to the β2 receptor, the constitutive internalization of the M3 receptor was largely unaffected by clathrin depletion whereas ligand stimulated internalization was inhibited (see below). These data show that GPCRs display constitutive and agonist-dependent internalization through clathrin-independent (CIE) and clathrin-dependent (CDE) endocytosis, respectively. Having demonstrated that β2 and M3 receptors constitutively internalize and recycle back to the PM along the CIE pathway, we decided to investigate if, over time, these receptors could reach late endosomal and lysosomal compartments for degradation. After 8 h of incubation in either the absence or presence of ligand at 37°, the β2 receptor localized to structures that labeled with Lamp1, a marker for lysosomes, indicating that this receptor could reach degradative compartments (supplemental Fig. S6). Similar observations were made for the M3 receptor (not shown). G Proteins (Gαs and Gαq) Associate with CIE Before and After Activation of the Cognate GPCR (β2R and M3R)—Most of the GPCR functions depend on the activation of specific heterotrimeric G proteins. Although GPCR ligand-dependent endocytosis has been studied extensively, much less is understood about the trafficking of G proteins. We analyzed the localization in HeLa cells of the endogenous G proteins Gαs and Gαq because they are activated by β2 and M3 receptors, respectively. It was shown previousl" @default.
• W2049587780 created "2016-06-24" @default.
• W2049587780 creator A5027347875 @default.
• W2049587780 creator A5049577856 @default.
• W2049587780 date "2009-02-01" @default.
• W2049587780 modified "2023-10-17" @default.
• W2049587780 title "Constitutive Internalization of G Protein-coupled Receptors and G Proteins via Clathrin-independent Endocytosis" @default.
• W2049587780 cites W1499460529 @default.
• W2049587780 cites W1521405901 @default.
• W2049587780 cites W1594024004 @default.
• W2049587780 cites W1603274157 @default.
• W2049587780 cites W1968323673 @default.
• W2049587780 cites W1969464978 @default.
• W2049587780 cites W1979233943 @default.
• W2049587780 cites W1983095317 @default.
• W2049587780 cites W1983203888 @default.
• W2049587780 cites W1993717649 @default.
• W2049587780 cites W1996453362 @default.
• W2049587780 cites W1999591184 @default.
• W2049587780 cites W2015398088 @default.
• W2049587780 cites W2015704490 @default.
• W2049587780 cites W2017633690 @default.
• W2049587780 cites W2031780396 @default.
• W2049587780 cites W2032444338 @default.
• W2049587780 cites W2036329205 @default.
• W2049587780 cites W2038422963 @default.
• W2049587780 cites W2039087375 @default.
• W2049587780 cites W2044775506 @default.
• W2049587780 cites W2045254177 @default.
• W2049587780 cites W2057496996 @default.
• W2049587780 cites W2058917109 @default.
• W2049587780 cites W2058930537 @default.
• W2049587780 cites W2060073882 @default.
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• W2049587780 cites W2077694135 @default.
• W2049587780 cites W2078482767 @default.
• W2049587780 cites W2085437402 @default.
• W2049587780 cites W2086459628 @default.
• W2049587780 cites W2094469376 @default.
• W2049587780 cites W2104527752 @default.
• W2049587780 cites W2105012896 @default.
• W2049587780 cites W2108696499 @default.
• W2049587780 cites W2117754393 @default.
• W2049587780 cites W2119398013 @default.
• W2049587780 cites W2122582811 @default.
• W2049587780 cites W2128685320 @default.
• W2049587780 cites W2135587043 @default.
• W2049587780 cites W2136094046 @default.
• W2049587780 cites W2137960852 @default.
• W2049587780 cites W2140538678 @default.
• W2049587780 cites W2151462825 @default.
• W2049587780 cites W2152568000 @default.
• W2049587780 cites W2157959080 @default.
• W2049587780 cites W2161582173 @default.
• W2049587780 cites W2163352150 @default.
• W2049587780 cites W2163909866 @default.
• W2049587780 cites W2167806590 @default.
• W2049587780 cites W2171756360 @default.
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