Distinct Clathrin-Coated Pits Sort Different G Protein-Coupled Receptor Cargo

Authors


Alastair W. Poole, a.poole@bris.ac.uk or Stuart J. Mundell, s.j.mundell@bris.ac.uk

Abstract

Upon activation, many G protein-coupled receptors (GPCRs) internalize by clathrin-mediated endocytosis and are subsequently sorted to undergo recycling or lysosomal degradation. Here we observe that sorting can take place much earlier than previously thought, by entry of different GPCRs into distinct populations of clathrin-coated pit (CCP). These distinct populations were revealed by analysis of two purinergic GPCRs, P2Y1 and P2Y12, which enter two populations of CCPs in a mutually exclusive manner. The mechanisms underlying early GPCR sorting involve differential kinase-dependent processes because internalization of P2Y12 is mediated by GPCR kinases (GRKs) and arrestin, whereas P2Y1 internalization is GRK- and arrestin-independent but requires protein kinase C. Importantly, the β2 adrenoceptor which also internalizes in a GRK-dependent manner also traffics exclusively to P2Y12-containing CCPs. Our data therefore reveal distinct populations of CCPs that sort GPCR cargo at the plasma membrane using different kinase-dependent mechanisms.

Following activation, many G protein-coupled receptors (GPCRs) undergo rapid clathrin-mediated endocytosis (1–3). This process mediates important functions for the cell, tuning its responsiveness to ligands over both short-term and long-term periods and regulating receptor coupling to signal transduction pathways. Endocytosis of GPCRs classically involves recruitment of agonist-occupied receptor into clathrin-coated pits (CCPs), which then form vesicles for entry into the endocytic pathway. At this early stage, the cell may decide to allow progression of vesicles and their cargo to further endosomal processing or may decide to recycle the cargo back to the plasma membrane. The molecular mechanisms underlying this decision are still not clearly defined but are receptor specific and may vary between cell types.

It is now clear that different GPCRs may utilize distinct pathways and interact with specific components of the endocytic trafficking machinery to regulate their internalization and recycling. Some GPCRs, including the β2-adrenoceptor rapidly internalize and recycle to the plasma membrane within the space of a few minutes (4–7). Many receptors, however, only slowly recycle to the cell surface after internalization, including the neurokinin NK1 receptor (8), αIB adrenoceptor (9) and the V2 vasopressin receptor (10–12). In part, the prolonged retention within the cell prior to recycling is due to high-affinity interaction with scaffolding partners, such as arrestin, binding to phosphorylated serine-rich sequences in the C-terminus of the receptor, such as is the case for V2 vasopressin receptor (10–12). The fate of internalized receptors may therefore partly be a consequence of their molecular interacting partners, but in other cases, their fate may be determined by the way in which they are internalized. For example, Vickery and von Zastrow (13) elegantly show that the highly related D1 and D2 dopamine receptors are processed quite differently as a consequence of their distinct internalization by dynamin- and clathrin-dependent (D1) or -independent (D2) internalization mechanisms.

Extracellular nucleotides potently trigger a broad range of physiological responses including cardiac muscle contraction, pain initiation and platelet activation. ADP plays a central role in platelet activation by acting as a released autocrine mediator in platelet responses to other agonists. ADP activates two platelet GPCRs, P2Y1 and P2Y12 (14,15), which couple to Gq- and Gi-mediated pathways, respectively, and synergize to achieve full platelet aggregation responses to ADP (14,16). We and others have recently shown that upon prolonged exposure to agonist, the responsiveness of both P2Y1 and P2Y12 receptors in human platelets decreases (17–19). This desensitization is mediated by differential kinase-dependent mechanisms, where P2Y1 responses desensitize in a protein kinase C (PKC)-dependent but GPCR kinase (GRK)-independent manner whereas P2Y12 responses desensitize in a PKC-independent but GRK-dependent manner (17). These two receptors therefore provide a useful model for comparative analysis of receptor internalization mechanisms.

Here we show that, when expressed in 1321N1 astrocytoma cells, both P2Y1 and P2Y12 internalize through clathrin-mediated endocytosis, although by differential kinase-dependent processes in line with their desensitization (17). Importantly, however, the two receptors sort to distinct membrane CCPs but rapidly colocalize at early endosomal vesicles. Both receptors recruit to CCPs using kinase-dependent processes; however, P2Y1 internalization depends upon PKC but not GRKs, whereas the converse is the case for P2Y12. These data suggest, for the first time, that distinct classes of CCP exist to sort GPCRs at the plasma membrane, using different kinase-dependent recruitment mechanisms to enable sorting at this early stage.

Results

P2Y1 and P2Y12 move to distinct endocytic loci on the plasma membrane but rapidly colocalize after internalization

We have recently shown that the platelet P2Y1 and P2Y12 are regulated differently and that the receptors desensitize by distinct kinase-dependent mechanisms where P2Y1 is PKC-dependent but GRK-independent, while P2Y12 is PKC-independent but GRK-dependent (17). Not only are these receptors of primary physiological importance for the haemostatic function of platelets but also their differential control by kinases pointed to potential differences in their internalization, intracellular trafficking and recycling to the plasma membrane. Receptors tagged with haemagglutinin (HA) were expressed in the P2Y null 1321N1 human astrocytoma cell line (20–23). Both P2Y1 and P2Y12 were primarily basally localized to the cell surface (Figure 1A) and underwent redistribution to a punctate pattern following stimulation with ADP (10 μm; 30 min). Figure 1B shows by enzyme-linked immunosorbent assay (ELISA) that there was a rapid ADP (10 μm)-induced internalization of both receptors reaching 30–40% loss of cell surface receptor by 15-min stimulation.

Figure 1.

Figure 1.

Agonist-induced internalization of P2Y1 and P2Y12 purinergic receptors. A) 1321N1 cells stably expressing either HA-tagged P2Y1 or P2Y12 receptor were preincubated with an anti-HA antibody at 4°C for 1 h. Subsequently, cells were incubated at 37°C for 30 min in the absence or presence of agonist (ADP; 10 μm). Receptor localization was determined by immunofluorescence in fixed cells and visualized using a fluorescein-conjugated secondary antibody. Data shown are representative of three independent experiments. B) 1321N1 cells stably expressing either HA-tagged P2Y1 or P2Y12 receptor were subsequently challenged with ADP (10 μm; 0–30 min). Surface receptor loss was subsequently assessed by ELISA as described in Materials and Methods. The scale bar represents 10 μm. The data represent means ± standard error of the mean (SEM) of five independent experiments.

In order more directly to compare endocytic processing of the two receptors, it was important to study the spatiotemporal dynamics of their internalization when coexpressed in the same cell. We therefore transiently transfected FLAG-tagged P2Y12 into 1321N1 cells stably expressing HA-P2Y1 and assessed the localization of each receptor following acute agonist stimulation (Figure 2A). Contributions from detection of FLAG (red) or HA (green) were determined by densitometry and plotted as shown. This analysis revealed an unexpected but critical finding that at the early time-point of stimulation (5 min), there was minimal colocalization of P2Y1 and P2Y12 at the cell membrane (Figure 2A; note from the scatter plot that there are two distinct clusters of membrane spots stained either predominantly red or predominantly green), suggesting that each receptor is sorted to distinct endocytic loci on the membrane surface. The analysis also revealed that, once internalized, the receptors colocalized, as displayed by the single large cluster of spots stained more equally for each receptor. By way of a control, when transiently transfected into 1321N1 cells stably expressing HA-P2Y12, FLAG-tagged P2Y12 colocalized at the cell membrane with the HA-tagged version of the receptor (Figure 2B). Also in order to control for possible differences in epitope detection, experiments were repeated by transiently transfecting myc-tagged P2Y12 into 1321N1 cells stably expressing HA-P2Y1 (Figure 2C). As for FLAG-tagged P2Y12 shown in Figure 2A, there was minimal colocalization of P2Y1 and P2Y12 at the cell membrane, although once internalized, the two receptors colocalized in endocytic compartments.

Figure 2.

Figure 2.

P2Y12 and P2Y1 receptors localize to different CCPs upon agonist stimulation. 1321N1 cells stably expressing either HA-tagged P2Y1 (A & C) or P2Y12 (B) receptor were transiently transfected with pcNEO-FLAG-P2Y12 (A and B; 5 μg DNA per 100-mm dish) or pcNEO-myc-P2Y12 (C; 5 μg DNA per 100-mm dish) and preincubated with monoclonal anti-HA (HA-11) antibody, polyclonal anti-FLAG (M2) or monoclonal anti-myc antibody (4A6) at 4°C for 1 h. Subsequently, cells were incubated at 37°C with ADP (10 μm; 5 min). Receptor localization was determined by immunofluorescence. Tagged receptors were visualized with goat anti-rabbit rhodamine-conjugated or goat anti-mouse fluorescein-conjugated secondary antibody, respectively. Agonist-induced accumulations of receptor at or near the cell membrane are indicated by arrows. The degree of HA-P2Y1–FLAG-P2Y12 (A), HA-P2Y12–FLAG-P2Y12 (B) or HA-P2Y1–myc-P2Y12 (C) colocalization following agonist addition can be seen (colocalization in yellow). The scale bar represents 10 μm. Enlarged examples of the cell membrane showing receptor localization are also shown, as indicated. Data shown are representative of three independent experiments. The right-hand panel shows graphical representation of 150 spots, taken from more than six cells from each of three independent experiments. Spots localized at the membrane are shown as filled circles, whereas spots localized intracellularly are shown as open circles.

Our data were therefore reminiscent of the only other report of coexpressed GPCRs moving to distinct plasma membrane loci upon activation, which showed a similar phenomenon for the dopamine D1 and D2 receptors (13). There were two important differences between our observations and those of Vickery and von Zastrow, however: (i) unlike D1 and D2 receptors, P2Y1 and P2Y12 receptors rapidly converged to appear in the same early endosomal compartments and (ii) D1 and D2 receptors were shown to internalize by dynamin-dependent and -independent mechanisms, respectively, suggesting that D2 receptors were likely to internalize by a clathrin-independent mechanism whereas D1 receptors were likely to be clathrin-dependent. It was therefore important to determine the dynamin- and clathrin-dependency of internalization of P2Y1 and P2Y12.

P2Y1 and P2Y12 receptors internalize by clathrin-mediated endocytosis

A major mechanism for internalization of GPCRs is through clathrin-mediated endocytosis. To determine the role of this pathway for P2Y1 and P2Y12, cells were transiently transfected with dominant negative mutant (DNM) forms (more than fivefold over endogenous levels) of eps-15 (EΔ95–295; eps-15-DNM) or with dynamin (K44A; dynamin-DNM). Eps-15-DNM blocks CCP formation (24), and we have used it in this regard extensively to block clathrin-dependent transferrin (data not shown) and GPCR (25–27) internalization. Dynamin-DNM is deficient in its ability to bind GTP and inhibits dynamin-mediated scission of clathrin-coated vesicles from the plasma membrane (28). Expression of either eps-15-DNM or dynamin-DNM strongly inhibited ADP-induced (10 μm; 30 min) P2Y1 and P2Y12 internalization (Figure 3A). Surface expression levels (in arbitrary absorbance units/mg cell protein) of both receptors, as assessed by ELISA, in the absence of agonist was comparable and unaffected by expression of either eps-15-DNM or dynamin-DNM [P2Y1 (control vector alone, 0.71 ± 0.14; eps-15-DNM, 0.66 ± 0.18; dynamin-DNM, 0.78 ± 0.14) and P2Y12 (control vector alone, 0.65 ± 0.13; eps-15-DNM, 0.61 ± 0.11; dynamin-DNM, 0.67 ± 0.19); mean ± standard error of the mean of five independent experiments]. Figure 3B shows that both P2Y1 and P2Y12 receptors physically associate with both clathrin heavy chain and α-adaptin, as shown by co-immunoprecipitation. The association is markedly enhanced upon receptor activation with ADP for 5 min. This finding is confirmed by colocalization studies (Figure 3C,D). Following acute stimulation with ADP (10 μm, 5 min), both P2Y1 and P2Y12 colocalized with both clathrin heavy chain and α-adaptin at the cell membrane. Additionally, supplementary data shown in Movies S1 and S2 show, by live-cell imaging, colocalization of both HA-P2Y12 and HA-P2Y1 with dsRed clathrin, respectively.

Figure 3.

Figure 3.

P2Y1 and P2Y12 receptors internalize by clathrin-dependent endocytosis. A) P2Y1- or P2Y12-expressing cells were transiently transfected with 5 μg of DNM forms of eps-15 (EΔ 95–295; eps-15-DNM), dynamin (K44A; dynamin-DNM) or vector (pcDNA3) alone. Cells were subsequently challenged with ADP (10 μm; 30 min) and surface receptor loss assessed by ELISA. The data represent means ± SEM of five independent experiments. *p < 0.05 compared with respective pcDNA3 vector-transfected controls (Mann–Whitney U-test). B) 1321N1 cells stably expressing HA-P2Y1 or HA-P2Y12 or vector (pcNEO) alone were stimulated with ADP (10 μm; 5 min) at 37°C. Reactions were stopped by addition of ice-cold lysis buffer and receptor was immunoprecipitated from cell lysates using an anti-HA antibody (HA-11) and association with either endogenous clathrin heavy chain (i) or α-adaptin (ii) assessed by immunoblotting. Whole-cell lysates (WCL) lanes are included as positive controls for detection by anti-clathrin or anti-α-adaptin antibodies. (C and D) 1321N1 cells stably expressing either HA-tagged P2Y1 or P2Y12 receptor were preincubated with fluorescein-conjugated anti-HA antibody at 4°C for 1 h. Subsequently cells were incubated at 37°C with ADP (10 μm; 5 min) and fixed. Following cell permeabilization, C) clathrin or D) α-adaptin localization was assessed using a clathrin heavy chain antibody or α-adaptin antibody, respectively, followed by a rhodamine-conjugated secondary antibody (shown in red). Agonist-induced accumulations of receptor (green/left column) at or near the cell membrane are indicated by arrows. The degree of receptor–clathrin (C) or receptor–α-adaptin (D) colocalization following agonist addition can be seen in the overlay column (colocalization in yellow). The scale bar represents 10 μm. Enlarged examples of the cell membrane showing colocalization data are also shown. Data shown are representative of three independent experiments.

P2Y1 and P2Y12 receptors are internalized by distinct protein-kinase-dependent mechanisms

GRKs 2 and 6 are endogenously expressed in 1321N1 cells and are critical for desensitization of P2Y12 (17). Expression of DNM-GRK2 or DNM-GRK6 selectively attenuated agonist-induced P2Y12 receptor internalization while that of P2Y1 was largely unaffected (Figure 4A) (29,30). Additionally, we reduced GRK2 and GRK6 expression using a previously characterized small interfering RNA (siRNA) approach (17). Figure 5D showed this approach to selectively reduce endogenous GRK expression by approximately 80%. Reductions of GRK levels in 1321N1 cells selectively attenuated P2Y12 versus P2Y1 agonist-induced internalization (Figure 4B). Interestingly, cotransfection of both GRK2 and GRK6 siRNAs provided a more substantial blockade of P2Y12 receptor internalization than either siRNA alone.

Figure 4.

Figure 4.

Role of protein kinases in agonist-induced internalization of P2Y1 and P2Y12 receptors. (A and B) P2Y1- or P2Y12-expressing cells were transiently transfected with A) 5 μg of either DNM-GRK2, DNM-GRK6 or vector (pcDNA3) alone or B) with scrambled, GRK2- or GRK6-specific siRNA constructs and used 2 days later. Cells were subsequently challenged with ADP (10 μm; 30 min) and surface receptor loss assessed by ELISA. The data are mean ± SEM of five independent experiments. *p < 0.05 compared with respective vector-transfected controls (Mann–Whitney U-test). C) P2Y1- or P2Y12-expressing cells were pretreated with the PKC inhibitor GF109203X (1 μm; 15 min) and subsequently challenged with ADP (10 μm; 30 min). Surface receptor loss was subsequently assessed by ELISA. The data represent means ± SEM of five independent experiments. *p < 0.05 compared with respective controls not treated with GF109203X (Mann–Whitney U-test).

Figure 5.

Figure 5.

P2Y12 but not P2Y1 receptor internalization is mediated by arrestin in a manner dependent upon GRK2 and GRK6. A) P2Y1- or P2Y12-expressing cells were transiently transfected with 5 μg of DNM forms of arrestin-2 (319–418; arrestin-DNM) or vector (pcDNA3) alone. Cells were subsequently challenged with ADP (10 μm; 30 min) and surface receptor loss assessed by ELISA. The data represent means ± SEM of five independent experiments. *p < 0.05 compared with respective pcDNA3 vector-transfected controls (Mann–Whitney U-test). B) Cells grown on poly-L-lysine coverslips were transiently transfected with 0.5 μg of peGFP-N1-arrestin-2-GFP. Prior to stimulation and viewing, coverslips were mounted in an imaging chamber at 37°C. The initial diffuse cytoplasmic distribution of arrestin-2-GFP is shown prior to agonist stimulation (0 second). ADP (10 μm) was added and the redistribution of arrestin-2 was monitored in real time. The images shown were collected before agonist addition (0) or 240 seconds after agonist addition. The scale bar represents 10 μm. Data shown are representative of three independent experiments. C) 1321N1 cells stably expressing HA-P2Y1 or HA-P2Y12 or vector (pcNEO) alone were stimulated with ADP (10 μm; 5 min). Receptor was immunoprecipitated (IP) from cell lysates using an anti-HA antibody (HA-11) and endogenous arrestin-2 association assessed by Western blotting (WB). As shown, arrestin-2 (arr-2)–receptor association (see arrow for arrestin band beneath heavy chain) was only found in cells expressing the P2Y12 purinergic receptor. Data shown are representative of three independent experiments. D) P2Y12 receptor was immunoprecipitated as outlined above and arrestin association (top panel; see arrow for arrestin band beneath heavy chain) subsequently assessed in cell lysates with reduced GRK expression (siRNA promoted reductions in GRK2 and/or GRK6 expression shown in middle and bottom panels, respectively). E) Densitometric analysis of arrestin-2–P2Y12 receptor co-immunoprecipitation data shown in (D). In these experiments, agonist-induced increases in arrestin-2 co-immunoprecipitation are assessed versus nonstimulated controls. The data are mean ± SEM of three independent experiments.

We had shown previously that PKC plays a major role in desensitization of P2Y1-mediated responses (17). Pretreatment with the non-isoform-selective PKC inhibitor GF109203X (1 μm; 15 min) markedly reduced ADP-induced P2Y1 receptor internalization (Figure 4C). In contrast, ADP-stimulated P2Y12 receptor internalization was unaffected by GF109203X pretreatment.

P2Y12 internalization is arrestin-dependent whereas P2Y1 internalization is arrestin-independent

It was important to address whether the receptors were differentially regulated by arrestins, and we addressed this by three approaches. First, overexpression of a DNM form of arrestin-2 (319–418; arrestin-DNM) selectively attenuated agonist-induced P2Y12 receptor internalization while that of P2Y1 was not significantly affected (Figure 5A). This arrestin-DNM competes with both arrestin-2 and arrestin-3 for clathrin binding (31–34). Second, as activation of many GPCRs leads to rapid recruitment of arrestins to the plasma membrane (35–37), we studied the redistribution of green fluorescent protein (GFP)-tagged arrestins (Figures 5B and S3). Prior to agonist stimulation, arrestin-2-GFP displayed a diffuse cytoplasmic distribution (Figure 5B). Following addition of 10 μm ADP, a rapid translocation of arrestin-2-GFP from cytosol to membrane was observed in P2Y12- but not P2Y1-expressing cells. Similar findings were obtained with arrestin-3-GFP (Figure S3), where colocalization at plasma membrane focal loci was also shown with P2Y12 but not P2Y1 by staining for receptor HA tag.

Third, using the HA epitope to immunoprecipitate the receptor, we demonstrated that endogenous arrestin-2 co-immunoprecipitates with P2Y12, but not P2Y1, and that the association increased upon agonist (ADP 10 μm; 5 min) addition (Figure 5C). Although a degree of basal association between P2Y12 and arrestin-2 was consistently observed, the nature of this physical association is not determined currently. Additionally, siRNA knock down of GRKs 2 and 6 shows that the interaction of arrestin-2 with P2Y12 depends upon the activities of these kinases (Figure 5D,E).

The β2-adrenoceptor moves to the same CCPs as P2Y12, but not P2Y1, upon receptor activation

Finally, it was important to determine whether early plasma membrane sorting of GPCRs occurred also for other GPCRs. The β2-adrenoceptor internalizes by clathrin-mediated endocytosis in a GRK- and arrestin-dependent process. FLAG-tagged β2-adrenoceptors were transiently transfected into 1321N1 cells stably expressing HA-P2Y12 or HA-P2Y1, and the localization of each receptor was assessed following acute agonist stimulation (5 min; Figure 6). This analysis revealed preferential colocalization of β2 receptors with P2Y12 but limited colocalization with P2Y1 at the cell membrane (as shown by two distinct clusters in the scatter plot), suggesting that sorting of GPCRs at this very early stage may be a widespread mechanism.

Figure 6.

Figure 6.

P2Y12 and β2-adrenoceptors colocalize at the plasma membrane upon agonist stimulation. 1321N1 cells stably expressing either the HA-tagged P2Y1 (A) or P2Y12 (B) receptor were transiently transfected with pcDNA3-FLAG-β2 adrenoceptor and preincubated with a monoclonal anti-HA (HA-11) antibody or polyclonal anti-FLAG (M2) at 4°C for 1 h. Subsequently, cells were incubated at 37°C for 5 min with ADP (10 μm) and isoproterenol (10 μm). Receptor localization was determined by immunofluorescence. Haemagglutinin-tagged or FLAG-tagged receptor was visualized with a goat anti-mouse rhodamine-conjugated or goat anti-rabbit fluorescein-conjugated secondary antibody, respectively. Agonist-induced accumulations of receptor at or near the cell membrane are indicated by arrows. The degree of HA-P2Y1–FLAG-β2 (A) and HA-P2Y12–FLAG-P2Y-β2 (B) colocalization following agonist addition can be seen (colocalization in yellow). The scale bar represents 10 μm. Data shown are representative of three independent experiments. The right-hand panel shows graphical representation of 150 spots, taken from more than six cells from each of three independent experiments. Spots localized at the membrane are shown as filled circles, whereas spots localized intracellularly are shown as open circles.

Discussion

Here, we report a critical finding that not all CCPs are uniform in their ability to recruit GPCR cargo and that there are different populations of CCPs that are able to target different GPCRs through different adaptor mechanisms. This novel cellular compartmentation was revealed through the differential handling of P2Y1 and P2Y12 receptors that internalize by clathrin-mediated endocytosis, although by distinct kinase-dependent processes. P2Y12 internalizes in a GRK- and arrestin-dependent manner, whereas P2Y1 internalizes in a GRK- and arrestin-independent manner requiring PKC. We propose classifying CCPs for GPCRs into two subtypes. P2Y12 receptors then sort to type 1 pits (CCP-1), as do β2-adrenoceptors, whereas P2Y1 sorts to type 2 pits (CCP-2). The two classes of pits are likely to be functionally distinct also, possibly modifying cargo differentially to mark them for distinct endocytic processing. Both P2Y12 and β2-adrenoceptors rapidly recycle to the plasma membrane, whereas P2Y1 receptors traffic to lysosomes predominantly, and this early receptor sorting may mark cargo for these processing decisions.

There has been only one report to date showing evidence for the existence of different classes of CCP. Cao et al. showed that a distinct subset of CCPs mediates internalization of GPCRs, as illustrated by the β2-adrenoceptor that entered a subpopulation of CCPs carrying transferrin cargo (38). The authors put forward an elegant model for specialization of endocytic membranes, suggesting that in addition to a divergent sorting process where cargo is sorted at the early endosomal level in several directions, there is also a convergent sorting where transferrin receptors and β2-adrenoceptors converge from subpopulations of CCPs into the same endocytic compartments. Our observations also favour a convergent model because following internalization, P2Y1 and P2Y12 converge into a common endocytic vesicle. The importance of our convergent model is, however, that we show for the first time that distinct CCPs are able to sort different GPCRs, which until now have been thought to enter a single uniform population of CCPs. The same group has also shown that GPCR sorting may occur at the membrane when they showed that dopamine D1 and D2 receptors sort to distinct loci in dynamin-dependent and -independent manners (13). By implication, however, this suggested that the D2 receptor is internalized by a non-clathrin-dependent mechanism and, therefore, that the early sorting of receptors is based upon clearly different endocytic mechanisms. This is quite different to the present report, however, where we show that the two receptors P2Y12 and P2Y1 each move to CCPs but distinct classes of these CCPs. Although it was possible that the receptors may exist already in different microdomains before coating (39), we have evidence that disruption of rafts with β-methyl cyclodextrin or with overexpression of dynamin-K44A DNM has no effect upon receptor distribution before agonist treatment (data not shown). This would suggest that the receptors genuinely differentially recruit to distinct CCPs upon activation. Additionally, it was possible that the receptors segregate to distinct pits through mechanisms partly based on homotypic positive co-operativity, where interaction between receptors of the same type increases the affinity of interaction for additional receptors of the same type. This would tend to exclude receptors of a different class from entering a pit where a growing number of receptors of one type are accumulating. This is unlikely, however, for P2Y12 as although it does not colocalize with P2Y1, it does colocalize with β2-adrenoceptors (Figure 6). It is still possible that the observed differential recruitment mechanisms to CCPs may confer a kinetic advantage for one type of receptor over another. For example, P2Y122-adrenoceptor interaction with arrestin may promote faster recruitment of these GPCRs to CCPs functionally indistinguishable from those containing the P2Y1 receptor. This kinetic advantage would not necessarily prevent the colocalization of the P2Y1 with the P2Y12 receptor but would make such an event unlikely, as an individual CCP would become saturated with whichever receptor entered this structure more rapidly. The analysis of recruitment suggests that the pits are, however, highly segregated, as there are very few pits where any major degree of colocalization of P2Y1 and P2Y12 exists (Figure 2A,C), tending to argue against a kinetic distinction between the two receptors, unless that kinetic distinction was large. Irrespective of mechanism, however, distinct recruitment of receptors is shown to occur, and it will be important to identify the functional consequences of this early sorting of GPCRs.

In the present study, we find the mechanisms underlying trafficking of P2Y1 and P2Y12 to be differentially controlled by two sets of kinases. Phosphorylation of GPCRs by GRKs or PKC can enhance receptor affinity for arrestin-2 and arrestin-3 (40); however, it is clear that in the case of P2Y1, although internalization is regulated by PKC, it is not mediated by arrestin binding. A number of GPCRs have been shown to internalize via CCPs in an arrestin-independent manner (41). For example, arrestin-independent clathrin-mediated endocytosis of M3 muscarinic receptors requires βγ subunit-dependent recruitment of tubulin, and α1B-adrenoceptors are endocytosed through a clathrin-mediated pathway by direct interaction of the receptor with the AP2 complex machinery (42). P2Y1 contains a number of putative PKC phosphorylation consensus sites, and a recent study examining the regulation of P2Y1 identified a specific residue, Thr339, in the C-terminus of this receptor phosphorylated by PKC that promoted P2Y1 desensitization (43). It will be important to identify the molecular mechanism by which P2Y1 internalizes in a clathrin-dependent but arrestin-independent manner in order to isolate the molecular differences between the two populations of CCPs revealed in the present study.

It is clear from the present study that both GRK2 and GRK6 may contribute to regulation of internalization of P2Y12. Reduced expression of either GRK2 or GRK6, using a siRNA approach, did not completely abolish arrestin–receptor interaction. However, near-complete blockade of arrestin–P2Y12 interaction was evident when expression of both GRKs was reduced, indicating that both GRK2 and GRK6 are able to promote arrestin–receptor association. There is therefore some functional redundancy between these two GRK isoforms. Relatively little is known about the specificity and functional roles of GRK-isoform-promoted arrestin–receptor association. Interestingly, it has recently been reported that specific GRK isoforms might play distinct roles in regulating the functional capabilities of receptor-bound arrestin, with GRK5/6 more able to induce arrestin-dependent extracellular-regulated kinase (ERK) activation than GRK2/3 (44,45). These same studies also showed that GRK2 rather than GRK5/6 was the major kinase responsible for AT1AR and V2 vasopressin receptor phosphorylation and accordingly responsible for the largest fraction of arrestin-2 recruitment and receptor internalization. This suggested a nonredundancy between GRK isoforms, in that each isoform plays a distinct functional role in the cell. This therefore contrasted with our studies where both GRK2 and GRK6 are equally able to promote P2Y12 receptor internalization.

It is intriguing to speculate as to the function of the early sorting of P2Y1 and P2Y12 into two distinct CCPs. The rapid colocalization of P2Y1 and P2Y12 after internalization indicates that the function of early sorting may be for early processing. A recent report from Baurand et al. (19) showed that P2Y12 is largely retained on the plasma membrane upon activation with ADP. These authors suggest that this may either be due to poor internalization of the receptor or due to rapid recycling of the receptor to the membrane after internalization. We have data to show that P2Y12, but not P2Y1, rapidly recycles to the plasma membrane after internalization (data not shown), and interestingly, the kinetics of recycling of P2Y12 is similar to those of the β2-adrenoceptor, which colocalizes to the same CCP (Figure 6). It is possible therefore that the two classes of CCP are functionally specialized to flag proteins either for rapid recycling (CCP-1) or for lysosomal degradation (CCP-2). Our mechanism is clearly distinct from, but may be complementary to, a recent report showing two classes of early endosome where one population is highly dynamic and mobile, maturing quickly into late endosomes, and the other population is more static and matures into late endosomes more slowly (46). This therefore represents another mechanism for sorting of cargo prior to entry into early endosomes, and our current work presents evidence that for GPCRs, this very early sorting may take place through distinct CCPs.

In conclusion, we show for the first time that GPCRs may be sorted at a very early stage to distinct populations of CCPs. This is revealed by the trafficking of purinergic receptors P2Y12 and P2Y1, which internalize in arrestin-dependent and arrestin-independent manners, respectively, to enter two classes of pit, CCP-1 and CCP-2. Receptors that enter CCP-1, such as P2Y12 and β2-adrenoceptor, rapidly recycle after internalization, and therefore we propose that this pit differs functionally from CCP-2, the pit into which P2Y1 traffics upon receptor activation and from which only slow GPCR recycling takes place. These observations reveal a greater degree of spatial sorting of GPCR cargo at the cell membrane than was previously thought, uncovering the existence of novel subpopulations of CCPs for distinct GPCRs.

Materials and methods

Materials

DMEM, Lipofectamine 2000 and FBS were obtained from Life Technologies Inc. (Invitrogen, Paisley, UK). Complete protease inhibitor tablets, fluorescein- and rhodamine-conjugated mouse monoclonal anti-HA antibody were from Roche (Roche Diagnostics Ltd, Lewes, East Sussex, UK). Anti-HA monoclonal antibody (HA-11) and goat anti-mouse fluorescein- or rhodamine-conjugated secondary antibody were purchased from Molecular Probes (Invitrogen). All other reagents were from Sigma (Sigma-Aldrich, Dorset, UK). DsRed clathrin was a kind gift from Dr Jim Keen, Thomas Jefferson University, Philadelphia, USA.

Construction of P2Y1 and P2Y12 receptor constructs

Haemagglutinin-tagged P2Y1 and P2Y12 constructs were generated by polymerase chain reaction (PCR) using the human P2Y1 and P2Y12 complementary DNAs as templates. Primers were designed which introduced a Xho restriction site at the 5′ end, followed by a Kozak sequence, an ATG and a nine-amino-acid HA tag (YPYDVPDYA) and an eight-amino-acid FLAG tag (DYKDDDDK). The 3′ primer sequence contained either an XbaI site (P2Y1) or a SalI site (P2Y12) to facilitate directional cloning of the PCR product into the pCMVneo vector (Promega, Southampton, UK). Sequencing of the final construct was performed to ensure that there were no mutations introduced during the PCR reaction.

Cell culture and transfection

1321N1 human astrocytoma cells were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin G and 100 μg/mL streptomycin sulphate at 37°C in a humidified atmosphere of 95% air and 5% CO2. Generation of stable transfectants of HA-P2Y1 and HA-P2Y12 using pCMVneo were as previously described (17). In transient transfections, cells were grown in 60- or 100-mm dishes to 80–90% confluence and transfected with DNA (amounts indicated in figure legends) using Lipofectamine 2000 according to the manufacturer's instructions. Cells were incubated with the DNA/Lipofectamine mixture for 24 h, the media replaced and cells analysed 48 h after transfection. Throughout the study, cell morphology was assessed to ensure that the cells were healthy and cell shape was comparable with other studies using these cells (47).

Transfection of GRK siRNAs

Small interfering RNA transfection of the GRK2, GRK6 and scrambled RNA duplexes (Dharmacon, Lafayette, CO, USA) was performed in 1321N1 cells at ∼90% confluence as previously described (17). Cells were transfected with siRNA duplexes (600 pmol siRNA in a 10-cm dish, final concentration of 40 nm) using Lipofectamine 2000. Cells were transfected with siRNA, split after 6 h, transfected a second time after 24 h and then analysed for GRK expression and receptor activity 4 days after initial transfection.

Internalization of proteins in 1321N1 cells assessed by ELISA

Haemagglutinin-tagged surface receptor loss was assessed by ELISA as described previously (36,48). Briefly, cells plated at a density of around 6 × 105 cells per 60-mm dish were transiently transfected with pcDNA3 containing GRK2, DNM-GRK2 (GRK2K220R), DNM-GRK6 (GRK6K215R) arrestin-2-DNM (arrestin-2 (319–418), 5 μg), eps-15-DNM (EΔ95–295) or dynamin-DNM (dynamin-K44A, 5 μg). Twenty-four hours post-transfection, cells were split into 24-well tissue culture dishes coated with 0.1 mg/mL poly-L-lysine. Twenty-four hours later, cells were incubated with DMEM containing apyrase (0.1 U/ mL) for 1 h at 37°C, washed and then challenged with DMEM containing ADP (10 μm) for 0–60 min at 37°C. Changes in surface receptor expression were subsequently determined by an immunosorbent assay (ELISA) taking advantage of the HA-epitope tag (36,48) and expressed as either % surface receptor or % loss of surface receptor with the background signal from pcDNA3-transfected controls subtracted from all receptor-transfected values.

Immunofluorescence microscopy of receptors in fixed 1321N1 cells

Cellular distribution of HA-tagged receptor or FLAG-tagged receptor in 1321N1 cells was assessed by immunofluorescence microscopy (36). Briefly, cells were grown on poly-L-lysine-coated coverslips in six-well plates. Twenty-four hours later, receptor distribution was assessed using a primary anti-HA-monoclonal antibody (HA-11; 1:200) and goat anti-mouse fluorescein-conjugated secondary antibody (1:200). Individual spots (intracellular or membrane) were identified and subsequently quantified (contribution from red and green channels, each assigned a densitometric measurement on a 0–255 scale) using Volocity software (Improvision, Coventry, UK).

Co-immunoprecipitation experiments

Following drug treatment and agonist stimulation at 37°C, 1321N1 cells from 100-mm dishes were washed twice with ice-cold PBS, lysed and co-immunoprecipitation experiments performed as previously described (26). Briefly, cells were lysed in ice-cold lysis buffer (1% Triton-X-100, 300 mm NaCl, 20 mm Tris, 1 mm phenylmethylsulphonyl fluoride and 10 mm ethylenediaminetetraacetic acid). Antibody–protein complexes were precipitated by incubation in the presence of anti-HA antibody (HA-11; 1:500) and protein A–agarose for 24 h at 4°C. Beads were washed twice with buffer before addition of 2× Laemmli sample solvent and boiling for 5 min. Proteins were detected by enhanced chemiluminescence. The extent of co-immunoprecipitation was quantified by densitometric analysis of resulting autoradiographs.

Experimental design and statistics

Data were analysed by the iterative fitting program GraphPAD Prism (GraphPAD Software, San Diego, CA, USA). Log concentration–effect curves were fitted to logistic expressions for single-site analysis, while t0.5 values for agonist-induced internalization were obtained by fitting data to single exponential curves. Where appropriate, statistical significance was assessed by Mann–Whitney U-test or by two-way anova.

Acknowledgments

We would like to thank Dr Suzanne Delaney, Portola Pharmaceuticals Inc., CA, USA, for valuable discussion of results in the preparation of this manuscript. We also thank Dr Jim Keen, Thomas Jefferson University, Philadelphia, USA for the kind donation of dsRed clathrin. A. R. H is supported by an A.J. Clark Studentship from the British Pharmacological Society. The work was supported by grants to A. W. P from the Wellcome Trust (grants 064785 and 069572) and the British Heart Foundation (grants PG/04/097/17620, FS/04/023, FS/05/017 and RG/05/015). S. J. M is a British Heart Foundation Research Fellow (grant no. FS/03/002/15102). The authors have no financial conflict of interest regarding work presented here.

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