Epitope-tagged Receptor Knock-in Mice Reveal That Differential Desensitization of α₂-Adrenergic Responses Is because of Ligand-selective Internalization^*

EXPERIMENTAL PROCEDURES

Generation of the HA-α_2AAR Knock-in Mice—An ADRA2A gene targeting vector was constructed with an HA sequence inserted at the 5′-end of the coding region and a neo resistance gene flanked by two loxP sites (cf. Fig. 1). The vector was introduced into sv129 embryonic stem cells via electroporation. Chimeric mice were generated by injection of targeted embryonic stem cells, and then crossed with C57Bl/6 mice to obtain F1 heterozygotes, which were further intercrossed to obtain homozygotes for verification of successful targeting. Electroporation and embryonic stem cell injection were performed by the Vanderbilt University Transgenic Core facility. To have the neo gene excised out of the targeted genome, F1 heterozygotes were crossed with CMV-cre mice (28) (backcrossed for 10 generations to C57Bl/6 background, generously provided by Dr. Mark Magnuson, Vanderbilt University). Cre recombinase catalyzes site-specific DNA recombination between the 34-bp recognition (loxP) sites, such as those with which we flanked the neo locus in our studies. In the strain of Cre mice used in our studies, the level of Cre expression is stable and sufficient to mediate deletion of any loxP-flanked locus in all cells of the body. Heterozygous knock-in mice encoding neither neo nor Cre genes were backcrossed to C57Bl/6 background for 10 generations and then intercrossed to generate homozygotes, which were fertile and developed normally.

Southern Analysis—Southern analysis was performed as described previously (29). DNA probes were labeled with [α-³²P]dCTP (PerkinElmer Life Sciences) using Prime-It II random primer labeling kit (Stratagene), and genomic DNA blots were hybridized using QuikHyb solution (Stratagene) following the manufacturer's instructions.

Radioligand Binding—Receptor density in WT and HA-α_2AAR knock-in mouse brains was evaluated by saturation binding of [³H]Rx821002 (Amersham Biosciences) as described previously (29). The intrinsic affinity of the α_2AAR for different ligands was assessed by competition binding of [³H]RX821002, as described (29, 30) in HEK cells stably expressing the murine HA-α_2AAR (31).

[³⁵S]GTPγS Binding—[³⁵S]GTPγS binding assays were performed as described previously (32, 33) to assess activation of G proteins by endogenous or knock-in HA-tagged α_2AAR in brain particulate preparations. WT or knock-in mouse brains were dissected and homogenized in membrane preparation buffer (50 mm Tris-HCl (pH 7.4), 1 mm EDTA, 3 mm MgCl₂). Particulate preparations were prepared by centrifugation of this homogenate at 20,000 × g for 20 min at 4 °C. After three washes in the same buffer, the particulate preparation was resuspended in assay buffer (50 mm Tris-HCl (pH 7.4), 100 mm NaCl, 3 mm MgCl₂, 0.2 mm EDTA) containing 10 μm GDP and 150 pm [³⁵S]GTPγS (1250 Ci/mmol, Amersham Biosciences). [³⁵S]GTPγS binding in response to the endogenous adrenergic agonist, epinephrine, was assessed in the presence of 1 μm prazosin (to block α₁, α_2B, and α_2CAR) and 1 μm propranolol (to block β-adrenergic receptors in the preparation). The percent stimulated [³⁵S]GTPγS binding was calculated by dividing each epinephrine-stimulated data point by the amount of signal for unstimulated (“basal”) [³⁵S]GTPγS binding. [³⁵S]GTPγS binding assays were also performed using HEK cells stably expressing the murine HA-α_2AAR (31), as described previously (30).

Localization of HA-α_2AAR in Brain and SCG Using Immunofluorescence—Adult male WT or HA-α_2AAR knock-in mice were transcardially perfused with phosphate-buffered saline, followed by 4% paraformaldehyde. Following post-fixation, 30 μm coronal sections of brain were sliced on a cryostat (Leica CM3050 S). Thirty micron sections of SCG were sliced on a microtome (Microm, HM400). Sections were then free-floated in wells of a 24-well plate for immunolabeling.

Sections were blocked with 4% normal donkey serum containing 0.2% Triton X-100 before incubation with primary antibody (mouse HA.11 antibody, Covance, 1:500-1:1000) for 48 h at 4 °C, followed by incubation with cyanine dye-conjugated secondary antibody (Cy2-conjugated donkey anti-mouse, Jackson ImmunoResearch, 1:1000) for 24 h at 4 °C. Sections were mounted on slides, sealed with PolyAquamount, and left overnight to dry. Images were taken with a Leica LSM-510 confocal microscope. Laser intensity was kept constant in comparing samples from the two genotypes.

Primary Culture of SCG Neurons—Primary cultures of SCG neurons were obtained from postnatal day 3-5 HA-α_2AAR knock-in mouse pups as described (34). In brief, SCG were dissected into L-15 medium (Invitrogen). After digestion with collagenase and trypsin (Sigma) in Hanks' balanced salt solution (Invitrogen), SCG were triturated with a polished Pasteur pipette, and cells were passed through a 40-μm strainer (Fisher). Following preplating on a noncoated culture dish to remove glial cells and fibroblasts, neurons were plated on coverslips pre-coated with poly-d-lysine and laminin (Sigma) in a 24-well plate in L-15 medium containing 10% NuSerum (Clontech), 30% glucose, 24 mm NaHCO₃, 2 mm glutamine, insulin/transferrin/sodium selenite media supplement (Sigma), and 25 ng/ml nerve growth factor (Invitrogen). Medium was changed every 3 days, with addition of 2 μm Ara-C (Sigma) to eliminate glial cell growth on the 2nd day. Immunofluorescence and electrophysiology experiments were performed on neurons cultured for 7-8 days in vitro, when α_2AAR was delivered to the cell surface (34).

Localization of WT-α_2AAR Versus HA-α_2AAR in Cultured SCG Neurons by Immunocytochemistry—SCG neurons were fixed in phosphate-buffered saline containing 3% sucrose and 3% paraformaldehyde (Electron Microscopy Sciences) for 15 min on ice and then permeabilized in PBST buffer (phosphate-buffered saline containing 0.1% Triton X-100) for 30 min at room temperature with the buffer changed every 10 min. Following blocking with 5% bovine serum albumin/PBST, neurons were incubated with antibodies against the C-terminal tail of endogenous α_2AAR (generously provided by Dr. Brian Kobilka, Stanford University) or HA.11 antibody (1-2 μg/ml) overnight at 4 °C. After washes, AlexaFluor 488-conjugated anti-rabbit and anti-mouse secondary antibodies (Invitrogen) were used to detect WT-α_2AAR and HA-α_2AAR, respectively. Images were acquired on a Leica confocal microscope with identical settings for both genotypes.

Quantitative Assessment of HA-α_2AAR Trafficking in Native SCG Neurons—Antibody labeling and quantitative fluorescent studies were performed as described previously (35). SCG neurons were first incubated with HA.11 antibody (10 μg/ml) for 12 min at room temperature to label cell surface HA-α_2AAR (no ligand-independent internalization was detected during the labeling). After washing off unbound antibodies, cells were treated with or without clonidine or guanfacine for various time periods followed by fixation and permeabilization. Although the α_2AAR is the major subtype expressed on the surface of SCGs cultured 7-8 days in vitro (34, 36), 10^-6 m prazosin was included in all trafficking experiments to ensure no potential activation of α_2B- or α_2CAR subtypes by clonidine or guanfacine. AlexaFluor 488-conjugated anti-mouse antibody (Invitrogen) was used to detect HA-α_2AAR labeled with HA.11 antibody. Images were obtained using a Leica confocal microscope, and all images were captured under identical microscope settings. Total and intracellular fluorescence intensities were quantified using MetaMorph software (Molecular Devices) as described previously (37). Relative units of internalization were measured as internal/total fluorescence normalized to untreated controls. Neurons from at least three independent experiments were analyzed.

Electrophysiology and Data Analysis—Ca²⁺ currents were recorded in cultured SCG neurons using standard whole-cell voltage clamp methods with an Axopatch 200B amplifier (Axon Instrument) as described previously (38, 39). No significant difference was observed between neurons cultured from HA-α_2AAR knock-in and WT mice in terms of Ca²⁺ current properties, acute response, and time course of Ca²⁺ current inhibition by different α₂-agonists. To correlate with the trafficking studies, the electrophysiological studies were performed in SCG neurons cultured from HA-α_2AAR knock-in mice. Cells were bathed in an external solution containing (in mm) 133 NaCl, 1 CaCl₂, 0.8 MgCl₂, 25 HEPES (pH 7.4), 12.5 NaOH, 5 glucose, 10 tetraethylammonium chloride, and 0.3 μm tetrodotoxin. Patch electrodes with resistances of 2-4 megohms were pulled from borosilicate glass and filled with an internal solution containing (in mm) 150 CsCl, 5 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, 5 Mg-ATP, and 10 HEPES (pH 7.2). Whole-cell Ca²⁺ currents were filtered at 1 kHz and digitized at 20 kHz. Data acquisition and analysis were performed with pCLAMP9 software.

The inhibition of Ca²⁺ currents induced by clonidine or guanfacine was measured using a two-pulse protocol as described previously (40, 41). The extent of inhibition was calculated as the difference in the peak current amplitude between the control current and that preceded by a brief depolarizing prepulse to 100 mV. To assess the time course and extent of desensitization of the clonidine- or guanfacine-evoked effect, inward currents were repetitively evoked every 20-40 s in the presence of clonidine or guanfacine. Recordings were made both in the presence and absence of prazosin (which blocks the α_2B- and α_2CAR subtypes). The presence of prazosin had no significant effect on the inhibition of Ca²⁺ currents by clonidine or guanfacine, confirming an α_2AAR-mediated process (42). The peak inhibition of Ca²⁺ currents by clonidine in the presence and absence of prazosin is 26.03 ± 1.85 (n = 15) and 30.07 ± 2.45 (n = 9), respectively, p = 0.26. The peak inhibition of Ca²⁺ currents by guanfacine in the presence and absence of prazosin is 24.42 ± 2.16 (n = 14) and 28.16 ± 2.51 (n = 9), respectively, p = 0.31. All recordings were normalized to Ca²⁺ rundown. To block receptor internalization, neurons were preincubated with ConA (160 μg/ml) or PAO (10 μm) for 20 min before recording and addition of ligands.

Intact Cell Receptor Phosphorylation—Intact cell phosphorylation of HA-α_2AAR in response to different agonists was performed as described previously (43) using HEK293 cells stably expressing murine HA-α_2AAR (31). Cells prelabeled with [³²P]orthophosphate (PerkinElmer Life Sciences) were stimulated for 20 min with or without clonidine or guanfacine, and cell lysates were subjected to immunoprecipitation using an anti-HA antibody. Immunoprecipitates were separated on 10% SDS-PAGE and analyzed by autoradiography (for phosphorylation) or Western analysis (for total HA-α_2AAR protein). To examine the effect of ConA on α_2AAR phosphorylation, cells were pretreated with 160 μg/ml ConA before agonist stimulation.

Statistical Analysis—Statistical analyses were performed with the GraphPad Prism software using Student's t tests. p < 0.05 was considered statistically significant.

RESULTS

Generation and Characterization of HA-α_2AAR Knock-in Mice—We utilized a homologous recombination gene targeting strategy to introduce the HA epitope-tagged wild type α_2AAR into the mouse ADRA2A gene locus (knock-in). The N-terminal HA epitope, which provides an extracellular “tag” readily detectable by commercial antibodies, has been widely used to tag GPCRs, including the α₂AR subtypes (44), without altering receptor trafficking and signaling. The gene targeting strategy is illustrated in Fig. 1A. Southern and PCR analyses of mouse tail DNA demonstrate successful generation of the final HA-α_2AAR knock-in mouse line (Fig. 1, B and C).

Expression density of HA-α_2AAR in the knock-in mice, which is controlled by the endogenous ADRA2A locus, was indistinguishable from that of the α_2AAR in WT mice, based on saturation binding analysis for the α_2AAR in particulate preparations from the brains of WT or HA-α_2AAR knock-in mice (Fig. 1D). Similarly, affinity for the endogenous ligand, epinephrine, was indistinguishable between the HA-α_2AAR in knock-in mice and α_2AAR in WT mice, as revealed by competition binding studies (Fig. 1E). Moreover, the addition of an N-terminal HA tag did not affect the ability of the HA-α_2AAR to activate G proteins in response to epinephrine in native tissues, as demonstrated by epinephrine-stimulated [³⁵S]GTPγS binding assays on brain particulate preparations from knock-in or WT mice (Fig. 1F). These data indicate that the knock-in HA-α_2AAR is expressed at the same density, shows identical affinity for the endogenous agonist, and exhibits indistinguishable intrinsic activity in activating endogenous G proteins as the α_2AAR in WT mice. Therefore, the HA-α_2AAR knock-in mouse provides an elegant model to examine α_2AAR trafficking and signaling in native cells expressing the endogenous receptor at its characteristic physiological density.

Distribution of Knock-in HA-α_2AAR in Mouse Brain and SCG—Using an HA antibody and a fluorescence-conjugated secondary antibody, we were able to examine the distribution of HA-α_2AAR in knock-in mouse brain slices. Fluorescent signals were readily detected in the lateral septum, locus coeruleus, and bed nucleus of the stria terminalis (Fig. 2A), as well as in the nucleus tractus solitarius and cortex (data not shown) of the knock-in brain, areas where α_2AAR mRNA transcription was previously reported (45). As expected, no positive signal for the HA epitope was detected in corresponding areas of the brain obtained from WT mice (Fig. 2A). Strong immunofluorescent signals also were detected in the SCG of knock-in mice. In the SCG, over 95% of the neurons are adrenergic (46) and labeled by an antibody against tyrosine hydroxylase (Fig. 2B). The HA-α_2AAR is mainly localized on the surface of neuronal cell bodies and neuronal fibers (Fig. 2B).

In cultured SCG neurons from knock-in mice, the distribution pattern of HA-α_2AAR (Fig. 2C) in the absence and presence of agonist stimulation is indistinguishable from that of the α_2AAR obtained from WT mice (Fig. 2D). WT α_2AAR was detected by an antibody directed against the C-terminal tail of the α_2AAR (34). These data confirm that the knock-in HA-α_2AAR can be reliably used for examining α_2AAR trafficking in native SCG neurons in parallel with our functional studies.

Clonidine and Guanfacine Differentially Promote α_2AAR Internalization in Primary Cultured SCG Neurons—We examined surface HA-α_2AAR trafficking in response to stimulation by clonidine or guanfacine in cultured SCG neurons. Following 5 min of treatment with 10^-6 m clonidine or guanfacine, internalization of cell surface HA-α_2AAR could be detected in response to clonidine but not to guanfacine (Fig. 3A). After 15 min of incubation with clonidine, a remarkable fraction of HA-α_2AAR had moved from the surface into cytosolic compartments and formed puncta in a perinuclear area (Fig. 3A). In contrast, only scattered puncta of HA-α_2AAR were observed inside cells following incubation with guanfacine for 15 min (Fig. 3A). The extent of internalization of cell surface HA-α_2AAR in response to clonidine was about 3.1-fold greater than that induced by guanfacine (Fig. 3B). A similar phenomenon was observed with 10^-5 m of clonidine and guanfacine (Fig. 3B). These data provide the first documentation that clonidine and guanfacine differentially evoke the trafficking of the endogenous α_2AAR in native sympathetic neurons.

Clonidine Induces Greater Phosphorylation of HA-α_2AAR than Guanfacine, Despite Comparable Receptor Affinity and Intrinsic Activity for These Two Ligands—To explore a possible molecular basis for the greater internalization of HA-α_2AAR in response to clonidine compared with guanfacine, we examined whether clonidine and guanfacine exhibit different binding affinities or intrinsic activities at the α_2AAR, under the same experimental settings. Fig. 4A demonstrates that the HA-α_2AAR has an indistinguishable affinity for clonidine and guanfacine, based on competition of these agents for [³H]RX821002 binding. Binding studies with the nonhydrolyzable GTP analogue, [³⁵S]GTPγS, indicate that these two agonists display a similar efficacy and potency in stimulating coupling of the occupied α_2AAR to G proteins (Fig. 4B). These data suggest that different abilities for inducing α_2AAR trafficking exhibited by clonidine and guanfacine cannot be attributed to receptor affinity or intrinsic activity of these two ligands at the α_2AAR.

We have previously found that receptor phosphorylation is required for arrestin-mediated internalization of the α_2AAR.5 Therefore, we examined the relative ability of these two agents to evoke α_2AAR phosphorylation using HEK293 cells stably expressing HA-α_2AAR. As shown in Fig. 4C, clonidine evokes a significantly higher level of α_2AAR phosphorylation than guanfacine at the same concentration of ligand (10^-6 m), which could subsequently lead to a stronger arrestin binding and receptor internalization. This result correlates with our observation in SCG neurons that clonidine induces a greater internalization than guanfacine.

Clonidine Evokes a Sustained and Greater Desensitization of Ca²⁺ Current Inhibition than Guanfacine in Cultured SCG Neurons—Because both receptor phosphorylation and internalization have been proposed as major mechanisms in regulating signaling response duration evoked by the receptor (2, 6), we postulated that the observed differential abilities of clonidine and guanfacine to promote α_2AAR phosphorylation and internalization might result in different desensitization profiles of α_2AAR-mediated signaling processes in response to these two drugs. To test this hypothesis, we evaluated α_2AAR-mediated inhibition of voltage-gated Ca²⁺ channel currents in SCG neurons using a two-pulse protocol (40) in response to incubation with clonidine versus guanfacine. Because desensitization of this response is highly dependent on agonist concentration (38), we chose to stimulate the receptor with 10^-5 m clonidine or guanfacine, the concentration that leads to maximum occupancy of the α_2AAR (Fig. 4A), to evaluate the maximal desensitization rate of α_2AAR-mediated inhibition of Ca²⁺ currents by these two agonists. Clonidine and guanfacine treatment induced a similar level of acute inhibition (∼30%) of Ca²⁺ currents (Fig. 5A). However, the sustained inhibitory effects of these two agents on Ca²⁺ currents were remarkably different. Specifically, inhibition of Ca²⁺ currents by clonidine began to wane after 200 s of incubation with this agonist, and dropped to ∼25% of the maximal inhibition level after 5 min of incubation (Fig. 5B), whereas inhibition of Ca²⁺ currents by guanfacine was maintained through the 5-min time point (Fig. 5C). After 15 min of exposure to the agonist, clonidine-evoked inhibition of Ca²⁺ currents continued to drop to nearly 50% of the maximal inhibition level (Fig. 5B), whereas only a slight decrease in Ca²⁺ current inhibition was observed in response to guanfacine (Fig. 5C). These data indicate that clonidine induces a faster and more dramatic desensitization of α_2AAR-evoked inhibition of Ca²⁺ currents than does guanfacine.

Blockade of Internalization Attenuates Desensitization of the Clonidine-induced Response but Has No Effect on Desensitization of the Guanfacine-induced Response—Next, we sought to directly address the causal relationship between greater receptor internalization and the more rapid and extensive desensitization of α_2AAR-mediated inhibition of Ca²⁺ currents induced by clonidine as compared with guanfacine. Therefore, we evaluated the desensitization of Ca²⁺ current inhibition evoked by 10^-5 m clonidine or guanfacine in the presence and absence of ConA treatment, a well established way to inhibit GPCR endocytosis in various cell types, including neurons (47-49). Preincubation with ConA blocked HA-α_2AAR internalization in SCG neurons induced by either clonidine or guanfacine (Fig. 6A). More interestingly, ConA treatment attenuated desensitization of clonidine-induced inhibition of Ca²⁺ currents at both the 5- and 15-min time points (Fig. 6B) without affecting the ability of clonidine to induce receptor phosphorylation (Fig. 6D). Similarly, another widely used endocytosis inhibitor, PAO (50-52), also reduced desensitization of clonidine-induced responses at these time points (Fig. 6B). In contrast, neither ConA nor PAO treatment affected desensitization by clonidine at 200 s (Fig. 6B). These data strongly suggest a critical and time-dependent contribution of receptor internalization on desensitization of clonidine-induced α_2AAR-mediated inhibition of Ca²⁺ currents. Although significant desensitization of guanfacine-evoked inhibition of Ca²⁺ currents is detected after 15 min of treatment, ConA blockade of α_2AAR internalization had no effect on this desensitization (Fig. 6C), suggesting that guanfacine-induced desensitization does not require α_2AAR internalization. Taken together, these data suggest that differential desensitization of the endogenous α_2AAR in native sympathetic neurons to clonidine versus guanfacine relies on the ability of clonidine to rapidly and markedly promote α_2AAR internalization.

DISCUSSION

HA-α_2AAR Knock-in Mouse Line Permits Assessment of Trafficking and Signaling Relationships in Native Target Cells—Our data reveal that N-terminal HA-tagged α_2AAR in a knock-in mouse fully mimics the WT receptor in terms of α_2AAR distribution (Fig. 2), receptor density, intrinsic receptor binding affinity, and G protein activation (Fig. 1). This is especially fortunate because two other previously reported GPCR knock-in lines, green fluorescent protein-tagged rhodopsin (53) and green fluorescent protein-tagged δ-opioid receptor (54), have been shown to exhibit altered receptor densities. Furthermore, our knock-in HA-α_2AAR manifests an agonist-evoked redistribution profile that is indistinguishable from the WT α_2AAR (Fig. 2). Thus, the HA-α_2AAR knock-in mouse line provides a unique and powerful tool to study the relationship between trafficking and signaling in the context of native target cells.

Ligand-selective Regulation of Receptor Endocytosis—It has been proposed that a GPCR has multiple active states, and each agonist can promote its own active receptor conformation(s), which leads to activation of a particular subset of responses, including receptor trafficking (8-11). Indeed, ligand-selective regulation of receptor endocytosis has been observed for a number of GPCRs, including μ- (55-60), κ- (61, 62), and δ-opioid receptors (63), parathyroid hormone receptors (64, 65), serotonin receptors (66-69), the P2Y receptor (70), the dopamine D₁ receptor (71), and the α_2AAR (72).

Ligand-induced receptor internalization also can vary depending on the receptor density and cell types where the receptor is expressed. For example, morphine promotes rapid endocytosis of μ-opioid receptor (MOR) in striatal neurons (73) but induces little internalization of MOR in hippocampal neurons (74, 75). Therefore, to understand the functional relevance of ligand-selective regulation of receptor trafficking, it is essential to study the trafficking of endogenously expressed receptors in native target cells.

Exploiting our HA-α_2AAR knock-in mice, we demonstrated that clonidine induces a more dramatic internalization of the α_2AAR than guanfacine in sympathetic neurons (Fig. 3), even though these two ligands exhibit similar receptor affinity (Fig. 4A) and intrinsic activity (Fig. 4B). We postulate that this difference in internalization is because of the ability of clonidine to stimulate greater α_2AAR phosphorylation than guanfacine (Fig. 4C), thus fostering an enhanced interaction of arrestin with the receptor (76), which leads to a more rapid and extensive α_2AAR internalization.

Relationship between Internalization and Desensitization—Although GPCR desensitization and internalization are two closely linked events (as both require GRK and arrestins), the causal relationship between these two events has been controversial. Early studies of β-adrenergic receptors in cultured 13N21 cells, for example, showed a rapid loss of response to isoproterenol that preceded the loss of the receptor from the surface (77). On the other hand, Lohse et al. (47) showed that receptor down-regulation, due to receptor degradation post-endocytosis, may contribute as much as 20-30% to the desensitization process of β₂AR-mediated signaling. Similarly, whereas failure of morphine to induce internalization was proposed as a possible explanation for the lack of MOR desensitization to this drug (78), morphine was found to promote desensitization without inducing significant MOR internalization (79, 80), and internalization was subsequently proposed as a mechanism for recovery from desensitization (80). Perhaps one explanation for the apparent discrepancy among all of these findings is the reliance in some studies on heterologous systems overexpressing the receptors under study. Recently, using a transgenic approach, Arttamangkul et al. (49) demonstrated that internalization of endogenously expressed MOR is not required for either the desensitization or the resensitization process in primary cultured locus coeruleus neurons, a finding that is entirely counter to previous findings for this receptor when evaluated in heterologous cells.

Our data provide definitive evidence for the requirement of rapid and extensive internalization of the α_2AAR to achieve maximal desensitization of α_2AAR-evoked inhibition of Ca²⁺ currents by clonidine in native sympathetic neurons (Fig. 6). However, the immediate desensitization (200 s) induced by clonidine is not affected by blockade of endocytosis with ConA (Fig. 6). Based on these data, we speculate that desensitization of the α_2AAR-evoked Ca²⁺ inhibition by clonidine likely involves two independent mechanisms as follows: an early rapid desensitization process at the cell surface, which may be due to receptor phosphorylation and related phosphorylation-dependent events, and a more extensive desensitization at later stage, which requires removal of receptor from the cell surface via internalization. In contrast, desensitization induced by guanfacine, which occurs to a considerably lesser extent than that induced by clonidine (Fig. 6), may occur mainly at the surface, because blockade of endocytosis had no effect on α_2AAR desensitization induced by guanfacine (Fig. 6). Whether surface-dependent desensitization of guanfacine-induced responses is mediated through phosphorylation by GRK or other second messenger-dependent kinases requires further investigation, and will require strategies that can distinguish desensitization of the receptor from desensitization of the voltage-gated Ca²⁺ channels, which also are highly regulated by second messenger-dependent protein kinases. However, the importance of our findings is that, in endogenous target cells, different agonist ligands at the same receptor differentially evoke receptor desensitization and do so by differential reliance on various mechanisms, rather than simply having different relative impacts on a single mechanism for desensitization.

Ligand-induced desensitization and endocytosis of GPCRs have been implicated as critical modulating mechanisms for in vivo response sensitivity, duration, and tolerance (3). Our finding that clonidine induces a faster and more dramatic desensitization of α_2AAR-mediated inhibition of voltage-gated Ca²⁺ channel currents than does guanfacine is likely of significance in vivo, because α_2AAR-mediated inhibition of norepinephrine release relies significantly on this signaling response (81). Our data provide a potential cellular mechanism explaining the longer duration of clinical efficacy of guanfacine compared with clonidine, namely clonidine-evoked acceleration of endocytosis and desensitization of the α_2AAR, which is temporally and quantitatively different from the effects of guanfacine.

Given the common existence of ligand-selective regulation of GPCRs, unveiling ligand-dependent regulation of trafficking and signaling of endogenous receptors in native target cells represents a powerful strategy for targeting the critical causal events in GPCR-involved pathophysiological responses, a first step in the strategic design of therapeutic intervention.