Clathrin box in G protein-coupled receptor kinase 2.

beta(1)-Adrenergic receptor (beta(1)AR) shows the resistance to agonist-induced internalization. However, beta(1)AR can internalize as G protein-coupled receptor kinase 2 (GRK2) is fused to its carboxyl terminus. Internalization of the beta(1)AR and GRK2 fusion protein (beta(1)AR/GRK2) is dependent on dynamin but independent of beta-arrestin and phosphorylation. The beta(1)AR/GRK2 fusion protein internalizes via clathrin-coated pits and is found to co-localize with the endosome that contains transferrin. The fusion proteins consisting of beta(1)AR and various portions of GRK2 reveal that the residues 498-502 in the carboxyl-terminal domain of GRK2 are critical to promote internalization of the fusion proteins. This domain contains a consensus sequence of a clathrin-binding motif defined as a clathrin box. In vitro binding assays show that the residues 498-502 of GRK2 bind the amino-terminal domain of clathrin heavy chain to almost the same extent as beta-arrestin1. The mutation of the clathrin box in the carboxyl-terminal domain of GRK2 results in the loss of the ability to promote internalization of the fusion protein. GRK2 activity increases and then decreases as the concentration of clathrin heavy chain increases. Taken together, these results imply that GRK2 contains a functional clathrin box and directly interacts with clathrin to modulate its function.

␤ 1 -Adrenergic receptor (␤ 1 AR) shows the resistance to agonist-induced internalization. However, ␤ 1 AR can internalize as G protein-coupled receptor kinase 2 (GRK2) is fused to its carboxyl terminus. Internalization of the ␤ 1 AR and GRK2 fusion protein (␤ 1 AR/GRK2) is dependent on dynamin but independent of ␤-arrestin and phosphorylation. The ␤ 1 AR/GRK2 fusion protein internalizes via clathrin-coated pits and is found to co-localize with the endosome that contains transferrin. The fusion proteins consisting of ␤ 1 AR and various portions of GRK2 reveal that the residues 498 -502 in the carboxyl-terminal domain of GRK2 are critical to promote internalization of the fusion proteins. This domain contains a consensus sequence of a clathrin-binding motif defined as a clathrin box. In vitro binding assays show that the residues 498 -502 of GRK2 bind the amino-terminal domain of clathrin heavy chain to almost the same extent as ␤-arrestin1. The mutation of the clathrin box in the carboxyl-terminal domain of GRK2 results in the loss of the ability to promote internalization of the fusion protein. GRK2 activity increases and then decreases as the concentration of clathrin heavy chain increases. Taken together, these results imply that GRK2 contains a functional clathrin box and directly interacts with clathrin to modulate its function.
Desensitization of G protein-coupled receptors (GPCRs) 1 plays an important role in the physiological regulation of receptor signal transduction. ␤ 2 AR that belongs to a GPCR family is recognized as a model system to delineate the molecular mechanism of desensitization that consists of three processes: uncoupling, internalization (endocytosis), and down-regulation (1). Within seconds to minutes after agonist stimulation, ␤ 2 AR is phosphorylated by GRKs and cAMP-dependent protein kinase. Subsequent binding of ␤-arrestins to the GRK-phospho-rylated ␤ 2 AR inhibits coupling of ␤ 2 AR with G s . The agonistbound ␤ 2 AR is also internalized from the plasma membrane to the intracellular endosome via clathrin-coated pits in a time period similar to that of uncoupling. Internalization is the process that contributes to functional resensitization by promoting dephosphorylation and recycling of the receptor to the plasma membrane (2,3). However, it has been reported that internalization contributes not only to uncoupling and recycling of receptors but also to down-regulation by promoting degradation of the receptors in lysosomes (4 -7).
Several studies have reported that GRKs and ␤-arrestins are implicated in internalization of many GPCRs. Overexpression of GRK2 enhanced internalization of m2AChR, whereas overexpression of a dominant-negative GRK2 inhibited it (8). Similarly, overexpression of GRK2 rescued agonist-promoted internalization of the internalization-defective ␤ 2 AR mutant (Y326A ␤ 2 AR), whereas the ␤ 2 AR mutant lacking putative GRK2 phosphorylation sites was not internalized (9). Overexpression of ␤-arrestin1 or ␤-arrestin2 rescued internalization of Y326A ␤ 2 AR, whereas dominant-negative ␤-arrestins inhibited internalization of WT-␤ 2 AR (10). These results show that GRK and ␤-arrestin play an essential role in internalization of some GPCRs. It has been reported that ␤-arrestins promote internalization by interacting not only with GPCRs but also with clathrin, the major component of the clathrin-based endocytic machinery (11). ␤-Arrestins bind clathrin by interaction of a consensus clathrin-binding motif in the carboxyl-terminal domain with the amino-terminal domain of the clathrin heavy chain (12).
The formation of vesicles for transport is mediated by coat proteins associated with integral membrane proteins (13). The clathrin-coated vesicle is the most characterized example, which consists mainly of clathrin and protein complexes termed adaptor protein-2 (AP-2) that forms at the plasma membrane when membrane receptors internalize (14). The regulatory proteins such as ␤-arrestins and amphiphysins, which have been demonstrated to interact with clathrin, have conserved sequences within the clathrin-binding regions (12,14). Alignment of these sequences defines a consensus motif for clathrin binding that consists of acidic and bulky hydrophobic residues and conforms to the canonical sequence L(L/I)(D/E/ N)(L/F)(D/E). This region can bind to the amino-terminal domain of clathrin heavy chain (16).
In contrast with ␤ 2 AR, ␤ 1 AR undergoes only slight internalization upon agonist stimulation (17). A lower affinity of ␤-arrestins for ␤ 1 AR than for ␤ 2 AR can explain the differential behavior of internalization between ␤ 1 AR and ␤ 2 AR. We determined the interaction of ␤-arrestins with ␤ 1 AR by several methods, and we found that ␤ 1 AR has a lower affinity for ␤-arrestins than ␤ 2 AR (18). In the course of analysis of the resistance of ␤ 1 AR to agonist-induced internalization, we have found that the ␤ 1 AR/GRK2 fusion protein internalized upon * This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture of Japan (to T. N. and H. K.). 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.
In the present study, we demonstrate that the carboxylterminal domain of GRK2 contains a consensus motif of clathrin binding shared with ␤-arrestins, amphiphysins, and AP-2. GRK2 directly interacts with clathrin via this clathrin-binding motif, and the phosphorylating ability of GRK2 is regulated by clathrin binding. It suggests that GRK2 participates in agonistpromoted internalization in more direct ways.
Plasmid Construction-The constructs of the HA-tagged ␤ 1 -and ␤ 2 ARs were described by Sato et al. (19). The ␤ 1 -and ␤ 2 AR/GRK2 fusion proteins were constructed as follows. First, various coding regions of GRK2 were amplified by two primers, which had restriction sites of SpeI or HindIII. The identity of the sequence of the PCR products was confirmed by thermal cycling sequencing kit from PerkinElmer Life Sciences. Then the entire coding region of GRK2 was ligated with ␤ 1 -or ␤ 2 ARs without a stop codon into EcoRI and HindIII of pRK5 by help of two oligonucleotides GATCCAGGAGGAGGAA and CTAGTTCCTC-CTCCTG. Because of these oligonucleotides, there are extra amino acids between GRK2 and each of receptors (Asp-Pro-Gly-Gly-Gly-Thr-Ser for ␤ 1 AR and Gly-Pro-Gly-Gly-Gly-Thr-Ser for ␤ 2 AR).
Expression and Purification of GST-␤-Arrestin1, GST-GRK2 Carboxyl-terminal Domain Fusion Proteins-The coding region of the carboxylterminal domain including stop codon of GRK2 (Asp 457 -Leu 689 ) was amplified and ligated into BamHI and SalI sites of pGEX-4T1. The entire coding region including stop codon of ␤-arrestin1 was amplified and ligated into EcoRI and SalI sites of pGEX-4T1. The resulting fusion protein constructs were used for transformation of BL21(DE3)pLys strain. The fusion proteins were then induced by the addition of 0.2 mM isopropyl-1-thio-␤-D-galactopyranoside for 24 h at room temperature (18,20). The GST fusion proteins were purified and concentrated by Centricon-30 as described (18).
Cell Culture and Transient Transfection of HEK293 Cells-HEK293 cells were cultured and transfected as described (18). Briefly, cells were cultured in DMEM supplemented with 10% fetal bovine serum and 10 g/ml gentamicin. The cells were seeded at density of 2 ϫ 10 5 cells for 6-well plates or 2 ϫ 10 6 cells for 100-mm dishes before the day of transfection. Then appropriate amounts of plasmids (2 g for 6-well plates or 8 g for 100-mm dishes) were used for transfection with 5 or 20 l of LipofectAMINE, according to the manufacturer's instructions. Cells were used for assays 2 days after transfection.
Internalization Assays-The binding activities of ␤ARs expressing in HEK293 cells were determined as described (18). Cells expressing ␤ARs in 6-well plates that had been transiently transfected were incubated at 37°C in serum-free DMEM with or without 10 M isoproterenol for the indicated time. Cells were washed three times by ice-cold phosphatebuffered saline and incubated for 3 h at 4°C with 6 nM [ 3 H]CGP-12177 in the modified KRH buffer (136 mM NaCl, 4.7 mM KCl, 1.25 mM MgSO 4 , 1.25 mM CaCl 2 , 20 mM HEPES, pH 7.4, 2 mg/ml bovine serum albumin). After incubation, cells were washed with ice-cold phosphatebuffered saline and then the cell-associated radioactivities were counted. Nonspecific binding was determined by the addition of 10 M propranolol. Inhibition of endocytosis was performed by maintaining cells in the medium (DMEM, 10% fetal bovine serum, and 0.5 M sucrose for hypertonic shock, 10 mM acetic acid for cytosol acidification or 0.25 mg/ml concanavalin A for cross-linking of lectin). Cells were washed once with one of the above mentioned medium and incubated for 30 min in the same medium. Then cells were stimulated by 10 M isoproterenol for 30 min. Inhibition of endocytosis was also performed by transient transfection with plasmids encoding dominant-negative dynamin (dynamin K44A) or dominant-negative ␤-arrestin2 (␤-arrestin2-(284 -409)). The expression levels of ␤ARs in the present study were 1080 Ϯ 103 fmol/mg (WT-␤ 1 AR), 742 Ϯ 93 fmol/mg (␤ 1 AR/GRK2), 694 Ϯ 51 fmol/mg (WT-␤ 2 AR), and 497 Ϯ 83 fmol/mg (␤ 2 AR/GRK2).
Phosphorylation of ␤ARs in HEK293 Cells-Phosphorylation of ␤ 2 AR, ␤ 1 AR, and the ␤ 1 AR/GRK2 fusion protein was performed with a slight modification of Kurose and Lefkowitz (21). HEK293 cells transiently expressing ␤ARs were labeled with 100 Ci/ml [ 32 P]PO 4 for 1 h in serum-and phosphate-free DMEM. The cells were then stimulated by 10 M isoproterenol for 10 min, and after extensive washing cells were scraped into buffer (10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 10 mM NaF, 10 mM sodium pyrophosphate) containing 10 g/ml benzamidine, 10 g/ml leupeptin, and 10 g/ml soybean trypsin inhibitor. The ␤ARs were solubilized by stirring for 1 h at 4°C and were centrifuged at 15,000 rpm for 30 min at 4°C. The solubilized ␤ARs were incubated with 2 g of anti-HA antibody and immunoprecipitated by incubating with protein A-Sepharose for 1 h. The immunoprecipitated receptors were separated from protein A-Sepharose by incubating them at 37°C for 30 min and resolved by 10% SDS-PAGE. The phosphorylated receptors were detected with Fuji BAS 1800.
Confocal Microscopy-Confocal microscopy was performed as described (18). HEK293 cells were transfected with the epitope-tagged and GRK2-fused ␤ 1 AR. Cells expressing the ␤ 1 AR/GRK2 fusion protein were plated onto 35-mm glass bottom dishes. On the day of experiment, the medium was replaced with serum-free DMEM containing 20 mM HEPES, pH 7.4. Cells were then incubated for 30 min with 40 g/ml Alexa Fluor TM 594-conjugated transferrin. After transferrin was endocytosed, cells were stimulated by 10 M isoproterenol for 30 min. Cells were washed, fixed with 4% formaldehyde, and permeabilized in 0.1% Triton X-100, and HA epitope at the amino terminus of the ␤ 1 AR/GRK2 fusion protein was detected by rat anti HA-antibody and fluorescein isothiocyanate-conjugated anti-rat antibody. Confocal images were obtained with two excitation wavelengths, 488 nm for ␤ 1 AR/GRK2 and 546 nm for transferrin. Signals were collected with a Laser Scanning Confocal Imaging System from Bio-Rad using Nikon CFI PLN APOC-HROMAT 60ϫ WI lens. The ␤ 1 AR/GRK2 was localized on the plasma membrane before agonist stimulation (data not shown).
Binding of in Vitro Translated Clathrin to GST Fusion Proteins-A cDNA fragment encoding residues 1-580 of human clathrin heavy chain was cloned by reverse transcriptase-polymerase chain reaction using Thermoscript TM and Pfx DNA polymerase according to the manufacturer's instructions. The PCR product was subcloned into BamHI and XhoI sites of the pBluescript SKII (Ϫ) vector and sequenced. The resulting amino-terminal domain of clathrin heavy chain was transcribed and translated in vitro using TNT quick-coupled transcription/ translation system in the presence of L-[ 35 S]methionine according to the manufacturer's instructions. The GST fusion proteins were immobilized on glutathione-Sepharose beads by incubating for 60 min at 4°C and then incubated for 60 min with in vitro translated clathrin in the binding buffer (50 mM Tris-HCl, pH 7.4, 100 mM KCl, 2 mM MgCl 2 , 0.1 mM CaCl 2 , 10% glycerol, 0.1% bovine serum albumin, 1% Triton X-100) at 4°C. After washing five times with the binding buffer, bound proteins were eluted and resolved by SDS-PAGE. The radiolabeled clathrin was detected by autoradiography and quantified by Fuji BAS 1800.
Purification of GRK2 and Phosphorylation of m2AChR-GRK2 and m2AChR were expressed in Sf9 cells with the baculovirus expression method (22). GRK2 eluted from the heparin column was stored at Ϫ80°C after the addition of a half-volume of glycerol. m2AChR was purified by affinity column chromatography and reconstituted into phospholipid vesicles as described. The amino-terminal region of clathrin heavy chain used for the in vitro ␤-arrestin binding experiment was inserted into pGEX-4T1. The resulting fusion protein was expressed and purified by the same way as GST-GRK2CT fusion proteins. Phosphorylation of m2AChR by GRK2 was performed by the incubation in buffer (10 M ATP, 20 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , 2 mM EDTA, 0.5 mM EGTA, 40 nM GRK2, 1 l of purified m2AChR, and 2.5 Ci of [␥-32 P]ATP) in the absence or presence of the indicated concentrations of GST-clathrin heavy chain or GST alone in a total volume of 30 l. The reaction was stopped after 10 min at 30°C. The reaction mixture was subjected to 10% SDS-PAGE, and the radioactive m2AChR was detected and quantified by Fuji BAS 1800.

Agonist-induced Internalization of ␤AR/GRK2
Fusion Proteins-We have determined agonist-induced internalization by measuring the numbers of WT-␤ARs or the ␤AR/GRK2 fusion proteins remaining at the cell surface. Consistent with the previous report (17,18,23), the number of ␤ 1 ARs was not decreased although the number of ␤ 2 ARs was decreased about 30% by isoproterenol stimulation for 30 min (Fig. 1). In contrast with WT-␤ 1 AR, the ␤ 1 AR/GRK2 fusion protein that GRK2 was fused to at the carboxyl terminus of ␤ 1 AR was internalized by isoproterenol stimulation (Fig. 1A). The ␤ 2 AR/GRK2 fusion protein internalized to a much greater extent than WT-␤ 2 AR (Fig. 1B). As receptor phosphorylation plays a central role in agonist-induced internalization (9), we determined the phosphorylation states of ␤ 2 AR, ␤ 1 AR, and ␤ 1 AR/GRK2 fusion protein. When ␤ 2 AR was expressed in HEK293 cells, ␤ 2 AR showed the increased phosphorylation by agonist stimulation (Fig. 2A). Under the same conditions, ␤ 1 AR was also phosphorylated by agonist stimulation, although the degree of phosphorylation was much smaller than that of ␤ 2 AR. In contrast to ␤ 1 AR, ␤ 1 AR/GRK2 fusion protein was efficiently phosphorylated by agonist stimulation (Fig. 2B). This result suggests that ␤ 1 AR has a lower affinity for GRK2 than ␤ 2 AR. It also raises the possibility that the enhanced internalization of the ␤ 1 AR/ GRK2 fusion protein is caused in part by the increased phosphorylation of ␤ 1 AR.
As the increased phosphorylation of the receptor recruits ␤-arrestins to promote internalization of the receptor, we determined whether the ␤ 1 AR/GRK2 fusion protein was internalized via ␤-arrestinand dynamin-dependent pathways. The carboxylterminal domains of ␤-arrestins interact with clathrin and AP-2 and promote internalization of the agonist-bound and -phosphorylated receptors (24). Therefore, the carboxyl-terminal domains of ␤-arrestins (for instance ␤-arrestin2-(284 -409)) can behave as dominant-negative mutants. Dynamin is a GTPase that pinches off the vesicles at the necks of clathrin-coated pits and is therefore essential for the formation of clathrin-coated vesicles (25,26). Therefore, dynamin mutating at Lys 44 that is important for GTP binding (dynamin K44A) works as a dominant-negative mutant. As shown in Fig. 3A, overexpression of ␤-arrestin2-(284 -409) and dynamin K44A significantly attenuated agonistpromoted internalization of ␤ 2 AR. In consistent with ␤ 2 AR, dynamin K44A completely prevented agonist-promoted internalization of the ␤ 1 AR/GRK2 fusion protein (Fig. 3B). However, ␤-arrestin2-(284 -409) was without effect on internalization of the ␤ 1 AR/GRK2 fusion protein. Consistent with the inability of ␤-arrestin2-(284 -409) to inhibit internalization, stimulation of the ␤ 1 AR/GRK2 fusion protein did not translocate ␤-arrestin2enhanced green fluorescent protein to the plasma membrane (data not shown). Internalization of the ␤ 1 AR/GRK2 fusion protein was not inhibited by a dominant negative ␤-arrestin2 mutant (␤-arrestin2-(284 -409)), and stimulation of the ␤ 1 AR/GRK2 fusion protein did not translocate ␤-arrestin2-enhanced green fluorescent protein to the plasma membrane. Therefore, it is concluded that the enhanced phosphorylation of the ␤ 1 AR/GRK2 fusion protein (shown in Fig. 2) does not result in the increased binding of ␤-arrestins. These results also suggested that the ␤ 1 AR/GRK2 fusion protein internalizes via a dynamin-dependent but ␤-arrestin-independent pathway. Next we examined the contribution of GRK2 fusion on the internalization behavior of ␤ 2 AR that efficiently internalizes upon agonist stimulation. The effect of GRK2-induced phosphorylation and ␤-arrestin binding on internalization was blocked by the expression of ␤-arrestin2-(284 -409). Therefore, we can determine the contribution of GRK2 fusion to internalization. ␤-Arrestin2-(284 -409) almost completely blocked internalization of ␤ 2 AR. However, ␤-arrestin2-(284 -409) partially inhibited internalization of the ␤ 2 AR/GRK2 fusion protein (Fig. 3C). This result suggests that GRK2 induces internalization by itself in a phosphorylationindependent manner when GRK2 is fused to the receptors. This result also indicates that the ␤ 2 AR/GRK2 fusion protein internalizes via at least two pathways. To examine whether internalization of the fusion protein is dependent on clathrin, internalization via clathrin-coated pits was blocked by various treatments (27,28). Stimulation with isoproterenol for 30 min promoted internalization of the ␤ 1 AR/GRK2 fusion protein by ϳ30% (Fig. 4). However, the incubation with 0.5 M sucrose, 0.25 mg/ml concanavalin A, or acetic acid resulted in the blockade of internalization of the ␤ 1 AR/GRK2 fusion protein. Taken together, these results indicate that ␤ 1 AR can be internalized when GRK2 was fused to the carboxyl terminus of ␤ 1 AR and that internalization of the fusion protein is dependent on clathrin and dynamin but independent of ␤-arrestin.
Visualization of Redistribution of Isoproterenol-stimulated ␤ 1 AR/GRK2 Fusion Protein-To determine the subcellular localization of the ␤ 1 AR/GRK2 fusion protein after agonist stimulation, we performed confocal microscopy to compare the localization of the fusion protein with that of transferrin, a well established marker of early and recycling endosomes (29,30). In the resting state, the fusion protein was localized at the plasma membrane and showed the different distributions from transferrin that was internalized and accumulated in early endosomes (data not shown). After agonist stimulation, the fusion protein appeared in endocytic vesicles that were randomly distributed throughout cytosol and co-localized with transferrin (Fig. 5, co-localization is shown by the appearance of the yellow color when the green color is coincident with the red color). This observation supports the idea that the ␤ 1 AR/ GRK2 fusion protein is really internalized by agonist stimulation and sorted to endocytic vesicles containing transferrin.
Internalization Behavior of Various Mutants of ␤ 1 AR/GRK2 Fusion Protein-To determine which region of GRK2 is responsible for differential behavior of agonist-promoted internalization of the fusion protein, we constructed various ␤ 1 AR/GRK2 mutants and determined the internalization behavior of them. The structures of ␤ 1 AR/GRK2 mutants are shown in Fig. 6A. Fig. 6B 4 and stimulated by 10 M isoproterenol. The receptors were solubilized, immunoprecipitated, and subjected to SDS-PAGE. The experiments were repeated three times, and similar results were obtained. The expression levels of ␤ARs were 59 Ϯ 12 fmol/mg (none), 370 Ϯ 120 fmol/mg (␤ 2 AR), 340 Ϯ 100 fmol/mg (␤ 1 AR), and 340 Ϯ 59 fmol/mg (␤ 1 AR/GRK2) of three experiments in duplicate. A, ␤ 2 AR but not ␤ 1 AR was strongly phosphorylated by isoproterenol (Iso) stimulation. B, the ␤ 1 AR/GRK2 fusion protein was also phosphorylated by isoproterenol stimulation, and no phosphorylation was observed in control HEK293 cells. ␤ 1 AR, these fusion proteins did not show agonist-promoted internalization (Fig. 6B). The ␤ 1 AR/GRK2 mutants that contained the carboxyl-terminal domain of GRK2 showed enhanced internalization, although the extent of internalization was not the same as that of ␤ 1 AR/GRK2. This result suggests that the carboxyl-terminal domain of GRK2 is necessary to promote internalization of the ␤ 1 AR/GRK2 fusion protein. After searching a motif for internalization in the carboxyl-terminal domain of GRK2, we found a potential clathrin-binding motif LLDSD at positions 498 -502. We suspected that direct interaction of the carboxyl-terminal domain of GRK2 with clathrin promoted internalization of ␤ 1 AR when the carboxylterminal domain of GRK2 was fused to ␤ 1 AR. Therefore, we mutated the amino acids in this region to alanine residues (Fig.  6A). When 498 LLDSD 502 in the carboxyl-terminal domain of GRK2 was mutated to alanine and fused to the ␤ 1 AR, this mutant did not internalize upon agonist stimulation (Fig. 6B). This result raised the possibility that 498 LLDSD 502 in GRK2 played an essential role in agonist-promoted internalization of the ␤ 1 AR/GRK2 fusion proteins by direct interaction with clathrin.
Interaction of in Vitro Translated Clathrin with Carboxyl Terminus of GRK2-The results in the previous sections indi-cate that GRK2 directly interacts with clathrin via its clathrinbinding motif in the carboxyl-terminal domain. It has been reported that the clathrin-binding motif binds to the aminoterminal domain of clathrin heavy chain. Therefore, we determined the ability of the carboxyl-terminal domain of GRK2 to bind clathrin. The GST fusion proteins of ␤-arrestin1 or the carboxyl-terminal domain of GRK2 were individually immobilized on glutathione-Sepharose beads and incubated with 35 Slabeled clathrin generated by in vitro translation. The bottom panel of Fig. 7A shows the Coomassie gel staining of GST fusion proteins that were used for the clathrin binding experiment. Although ␤-arrestin1-GST, GRK2CT-GST, and GRK2CT(AAAAA)-GST fusion proteins showed some degradation or truncation products, major bands of these fusion proteins were products of full-length inserts based on molecular weights of these products. Clathrin did not bind to GST alone (upper panel of Fig. 7A). In contrast with GST alone, clathrin bound strongly to ␤-arrestin1, consistent with the previous report (16). Clathrin also strongly bound to the carboxylterminal domain of GRK2. When the amino acids of clathrinbinding motif in the carboxyl-terminal domain of GRK2 were mutated to alanine (GRK2CT-498 LLDSD 502 3 GRK2CT-498 AAAAA 502 ), the clathrin binding to the carboxyl-terminal domain was greatly decreased (Fig. 7B). Considering the integrity and amounts of three fusion proteins (Fig. 7A), it is concluded that GRK2CT binds clathrin almost same degree as ␤-arrestin1, and alanine-substituted GRK2CT mutant binds much less than the other two fusion proteins. These results clearly demonstrate that GRK2 has a clathrin-binding motif in its carboxyl-terminal domain and interacts with clathrin heavy chain through that motif.
Regulation of GRK2-mediated m2AChR Phosphorylation by Clathrin-It has been reported very recently that class II PI3K-C2␣ is activated by clathrin (31). Therefore, we determined whether interaction of GRK2 with clathrin resulted in the enhanced phosphorylation activity of GRK2 toward agonist-bound receptor. As GRK2 can phosphorylate not only ␤ 2 AR but also m2AChR in vitro and in vivo, m2mAChR was used for in vitro phosphorylation experiments by GRK2. The fusion protein of clathrin heavy chain residues 1-580 with GST was expressed in Escherichia coli and purified from it. The purified preparation was not a full-length product (Fig. 8A). Because it has been reported that the amino-terminal portion is essential for interaction with clathrin-binding proteins (31), we used this fusion protein for the functional experiment. GRK2 phosphorylated agonist-bound m2AChR to some extent in the absence of any activator (Fig. 8B). The phosphorylation activity of GRK2 increased and then decreased as the concentration of the clathrin-GST fusion protein increased. This result indicates that clathrin modulates the function of GRK2. DISCUSSION In the present study, we demonstrated that GRK2 contains a functional clathrin binding domain. Our results provide GRK2 with a new function other than phosphorylation activity toward activated GPCRs. Many GPCRs are phosphorylated by GRKs in an agonist-dependent manner as a major mechanism of receptor regulation. There are six members of the GRK family (32). GRKs belong to the serine/threonine kinase family and show the property to phosphorylate agonist-occupied receptors, thereby triggering desensitization. GRK2 is the primary GRK expressed in the heart and is studied most extensively to date. Phosphorylation of GPCRs by GRK2 and subsequent binding of ␤-arrestins is one of the mechanisms leading to uncoupling from G proteins. The binding of ␤-arrestins to almost all GPCRs appears to enhance internalization through interaction of ␤-arrestins with clathrin and AP-2 (11,24). The vesicles for internalization consist of several proteins, which formed at the plasma membrane. The most extensively characterized proteins in the vesicles are clathrin and the protein complex termed AP-2 (33,34). Clathrin is the complex of three heavy chains and three light chains that polymerizes to form the cargo for internalization. AP-2 recruits the internalizing membrane proteins to the coated pits and also mediates attachment of clathrin to the membrane. ␤-Arrestins (12,16) as well as other proteins mentioned in Fig. 9 have been demonstrated to interact with clathrin. These clathrin-interacting proteins have conserved amino acid sequences for clathrin binding. This clathrin-binding motif is defined as a clathrin box that conforms to the canonical sequence L(L/I)(D/E/N)(L/ F)(D/E) (Fig. 9). In the present study, we found that GRK2 also has the sequence 498 LLDSD 502 similar to the canonical sequence of clathrin box in its carboxyl-terminal domain. As AP-2 and ␤-arrestins bind to the amino-terminal domain of clathrin heavy chain (17,24), we determined whether GRK2 also binds to the amino-terminal domain of clathrin heavy chain. We found that the carboxyl-terminal domain of GRK2 bound clathrin essentially the same extent to that of ␤-arrestin1. These results suggest that clathrin box of GRK2 is functional not only in binding of clathrin but also in enhancement of agonistpromoted internalization.
GRK2 is divided into three domains as follows: amino-terminal domain, catalytic domain, and carboxyl-terminal regula-tory domain. The amino-terminal domain contains regulators of G protein signaling (RGS) domains specific for G␣ q , and the carboxyl-terminal domain contains G␤␥ binding domain and pleckstrin homology (PH) domain that binds phosphatidylinositol 4,5-diphosphate (35,36). The present study adds another functional domain to GRK2, which interacts with clathrin and helps agonist-promoted internalization.
GPCRs internalize via several pathways. The most characterized and established internalization is the ␤-arrestinand dynamin-dependent pathway such as ␤ 2 AR internalizes. GRKmediated phosphorylation triggers internalization by promoting the binding of ␤-arrestins to the phosphorylated receptors and subsequent interaction with clathrin and AP-2. On the contrary, some GPCRs such as angiotensin II receptor and m2AChR internalize via ␤-arrestinand dynamin-independent pathway. In contrast to these GPCRs, m1, m3, and m4AChRs internalize via ␤-arrestin-independent but dynamin-dependent pathway (37,38). Direct interaction of GRK2 with clathrin may be involved in internalization of these mAChRs via dynamindependent pathway.
Affinity of substance P receptor for GRK2 seems to be higher than that for other GPCRs (39), because activation of substance P receptor but not other types of GPCRs including ␤ 2 AR and angiotensin II receptor induces translocation of GRK2 to the plasma membrane. A plausible explanation for this intriguing observation is that substance P receptor has higher affinity for GRK2 than other GPCRs (39). Therefore, GRK2 translocating to the plasma membrane may directly involve internalization of substance P receptor by interacting with clathrin.
Gaidarov et al. (31) reported very recently that clathrin activates the class II PI3K-C2␣ and regulates membrane trafficking via PI3K-C2␣. PI3K is classified into two classes, class I and class II. The class I PI3K is involved in postendocytic trafficking of the receptors and is sensitive to wortmannin and LY294002. PI3K-C2␣ belongs to class II PI3K and shows the resistance to PI3K inhibitors. Because PI3K-C2␣ is activated by clathrin and the consequent production of PIP 3 can recruit various PIP 3 -binding proteins, it was proposed that clathrin helps to stimulate endocytosis by the increased formation of PIP 3 (31). In the present study, we demonstrated that the amino-terminal domain of clathrin heavy chain binds the carboxyl-terminal region of GRK2 and modulates GRK2 phosphorylation activity. GRK2 activity was regulated by clathrin in a bell-shaped manner. Low concentration of clathrin-GST fusion protein increased, and high concentration decreased GRK2 phosphorylation activ- ity. The degree of activation by clathrin-GST fusion protein was lower than that of PI3K-C2␣ by clathrin complex. One of the possibilities to explain this low activation is that other parts of clathrin help to activate GRK2 upon interaction with GRK2. Another possibility is that the observed GRK2 activity reflects the combined effects of stimulatory and inhibitory regulation by clathrin even at a stimulatory phase. It is not apparent at present which phase (stimulatory or inhibitory) is physiologically important for GRK2-mediated receptor internalization. However, it has been reported that GRK2 phosphorylates a cytoskeletal protein, tubulin, and modulates its function (15). Therefore, it is interesting to speculate that activation of GRK2 by clathrin results in the enhanced phosphorylation of tubulin and leads to cytoskeletal rearrangement for internalization. Further studies will be necessary for resolving these issues. Our findings imply that GRK2 not only phosphorylates the receptor in an active conformation but also is likely to function as a clathrin adaptor, just like ␤-arrestins and AP-2. GRK2 may be more directly involved in the sorting events than previously envisioned.
In conclusion, we demonstrated that GRK2 directly interacts with clathrin via the clathrin box located at the carboxylterminal domain of GRK2. The clathrin box is a consensus motif shared with AP-2, ␤-arrestins, and amphiphysins to interact with the amino-terminal domain of clathrin heavy chain. Conservation of this motif is likely to account for the ability of all these proteins to bind clathrin despite their structural diversity. Therefore, ␤ 1 AR could internalize upon agonist stimulation when GRK2 was fused to the carboxyl terminus of ␤ 1 AR. FIG. 9. The sequence alignment of the clathrin box in various proteins involved in internalization. Sequence alignment illustrating clathrin box in proteins known to interact with clathrin is shown. It is believed that these proteins bind to the amino-terminal domain of clathrin. GRK2 also has this potential clathrin-binding motif at residues 498 -502.