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

Although ligand-selective regulation of G protein-coupled receptor-mediated signaling and trafficking are well documented, little is known about whether ligand-selective effects occur on endogenous receptors or whether such effects modify the signaling response in physiologically relevant cells. Using a gene targeting approach, we generated a knock-in mouse line, in which N-terminal hemagglutinin epitope-tagged α2A-adrenergic receptor (AR) expression was driven by the endogenous mouse α2AAR gene locus. Exploiting this mouse line, we evaluated α2AAR trafficking and α2AAR-mediated inhibition of Ca2+ currents in native sympathetic neurons in response to clonidine and guanfacine, two drugs used for treatment of hypertension, attention deficit and hyperactivity disorder, and enhancement of analgesia through actions on the α2AAR subtype. We discovered a more rapid desensitization of Ca2+ current suppression by clonidine than guanfacine, which paralleled a more marked receptor phosphorylation and endocytosis of α2AAR evoked by clonidine than by guanfacine. Clonidine-induced α2AAR desensitization, but not receptor phosphorylation, was attenuated by blockade of endocytosis with concanavalin A, indicating a critical role for internalization of α2AAR in desensitization to this ligand. Our data on endogenous receptor-mediated signaling and trafficking in native cells reveal not only differential regulation of G protein-coupled receptor endocytosis by different ligands, but also a differential contribution of receptor endocytosis to signaling desensitization. Taken together, our data suggest that these HA-α2AAR knock-in mice will serve as an important model in developing ligands to favor endocytosis or nonendocytosis of receptors, depending on the target cell and pathophysiology being addressed.

G protein-coupled receptors (GPCRs) 4 represent the largest family of cell surface receptors mediating responses to hormones, cytokines, neurotransmitters, and therapeutic agents (1). In addition to regulating downstream signaling, ligand binding to a receptor can initiate phosphorylation of the active conformation of the receptor by G protein receptor kinases (GRKs) and subsequent binding of arrestins, thus restricting the magnitude and duration of the ligand-evoked signaling responses (2,3). Binding of arrestins to GPCRs also leads to GPCR internalization (4,5), a process that has been proposed as a means to desensitize receptor signaling at the cell surface, resensitize receptors, and/or initiate intracellular signaling (6,7). Different ligands are able to induce distinct signaling and internalization profiles of the same receptor (8 -14). However, the lack of available tools to study trafficking of endogenous GPCRs in native target cells has limited our understanding of ligand-selective endocytosis profiles and the relative contribution of receptor endocytosis to desensitization in native biological settings.
To specifically test hypotheses regarding ligand-selective effects on GPCR internalization, and functional consequences of this trafficking on signaling, we utilized a homologous recombination gene targeting strategy to introduce a hemagglutinin (HA) epitope-tagged wild type ␣ 2A -adrenergic receptor (AR) into the mouse ADRA2A gene locus ("knock-in"). The ␣ 2A AR is a prototypical GPCR that couples to the G i/o subfamily of G proteins (15). Studies on genetically engineered mice made null or mutant for the ␣ 2A AR have revealed that this subtype mediates the therapeutic effects of ␣ 2 -adrenergic agents on blood pressure, pain perception, volatile anesthetic sparing, analgesia, and working memory enhancement (16 -18). Two classic ␣ 2 -ligands, clonidine and guanfacine, have been widely used to treat hypertension (19), attention deficit and hyperactivity disorder (20), and to elicit analgesia (19,21) mediated via the ␣ 2A AR. Clinically guanfacine has a much longer duration of action than clonidine (22)(23)(24); this longer duration of action cannot be accounted for by the pharmacokinetic profile of these agents in human beings, as both drugs have a half-life of 12-14 h (25, 26). Because ligand-induced desensitization and trafficking of GPCRs have been implicated as critical mechanisms for modulating response duration in vivo (3), one hypothesis underlying the difference in duration between clonidine and guanfacine is that clonidine provokes accelerated desensitization of the ␣ 2A AR via one or several mechanisms, whereas guanfacine does not. Signaling desensitization in response to these two agonists has not been compared under the same experimental settings. To specifically test this hypothesis, we have exploited our HA-␣ 2A AR knock-in mice so that we could examine these properties of guanfacine and clonidine in native target cells.
We compared internalization of the ␣ 2A AR and inhibition of Ca 2ϩ currents induced by clonidine and guanfacine in primary superior cervical ganglia (SCG) neurons, where the ␣ 2A AR is the major adrenergic receptor subtype controlling norepinephrine release and sympathetic tone (17,27). Our data revealed a differential regulation of ␣ 2A AR trafficking and signaling duration by clonidine versus guanfacine, i.e. clonidine induced a more dramatic desensitization of the ␣ 2A AR than guanfacine, and this desensitization was largely because of ␣ 2A AR internalization. These studies reveal the powerful tool that the HA-␣ 2A AR knock-in mice provide for identifying endocytosisdependent and -independent physiological phenomena for this receptor subtype as a first step in defining novel loci for therapeutic intervention in the ␣ 2A AR signaling/trafficking cascade.

EXPERIMENTAL PROCEDURES
Generation of the HA-␣ 2A AR Knock-in Mice-An ADRA2A gene targeting vector was constructed with an HA sequence inserted at the 5Ј-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 [␣-32 P]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-␣ 2A AR knock-in mouse brains was evaluated by saturation binding of [ 3 H]Rx821002 (Amersham Biosciences) as described previously (29). The intrinsic affinity of the ␣ 2A AR for different ligands was assessed by competition binding of [ 3 H]RX821002, as described (29,30) in HEK cells stably expressing the murine HA-␣ 2A AR (31).
[ 35 S]GTP␥S Binding-[ 35 S]GTP␥S binding assays were performed as described previously (32,33) to assess activation of G proteins by endogenous or knock-in HA-tagged ␣ 2A AR 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 MgCl 2 ). Particulate preparations were prepared by centrifugation of this homogenate at 20,000 ϫ g for 20 min at 4°C. After three washes in the same buffer, the particulate preparation was resuspended in assay buffer (50 mM  S]GTP␥S binding assays were also performed using HEK cells stably expressing the murine HA-␣ 2A AR (31), as described previously (30).
Localization of HA-␣ 2A AR in Brain and SCG Using Immunofluorescence-Adult male WT or HA-␣ 2A AR knock-in mice were transcardially perfused with phosphate-buffered saline, followed by 4% paraformaldehyde. Following post-fixation, 30 m 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 freefloated 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-␣ 2A AR 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-m 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 NaHCO 3 , 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 M 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 ␣ 2A AR was delivered to the cell surface (34).
Localization of WT-␣ 2A AR Versus HA-␣ 2A AR 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 (phosphatebuffered 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 ␣ 2A AR (generously provided by Dr. Brian Kobilka, Stanford University) or HA.11 antibody (1-2 g/ml) overnight at 4°C. After washes, AlexaFluor 488-conjugated anti-rabbit and anti-mouse secondary antibodies (Invitrogen) were used to detect WT-␣ 2A AR and HA-␣ 2A AR, respectively. Images were acquired on a Leica confocal microscope with identical settings for both genotypes.
Quantitative Assessment of HA-␣ 2A AR 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 g/ml) for 12 min at room temperature to label cell surface HA-␣ 2A AR (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 ␣ 2A AR is the major subtype expressed on the surface of SCGs cultured 7-8 days in vitro (34,36), 10 Ϫ6 M prazosin was included in all trafficking experiments to ensure no potential activation of ␣ 2B -or ␣ 2C AR subtypes by clonidine or guanfacine. AlexaFluor 488-conjugated anti-mouse antibody (Invitrogen) was used to detect HA-␣ 2A AR 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-Ca 2ϩ 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-␣ 2A AR knock-in and WT mice in terms of Ca 2ϩ current properties, acute response, and time course of Ca 2ϩ current inhibition by different ␣ 2 -agonists. To correlate with the traf-ficking studies, the electrophysiological studies were performed in SCG neurons cultured from HA-␣ 2A AR knock-in mice. Cells were bathed in an external solution containing (in mM) 133 NaCl, 1 CaCl 2 , 0.8 MgCl 2 , 25 HEPES (pH 7.4), 12.5 NaOH, 5 glucose, 10 tetraethylammonium chloride, and 0.3 M 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,NЈ,NЈ-tetraacetic acid, 5 Mg-ATP, and 10 HEPES (pH 7.2). Whole-cell Ca 2ϩ currents were filtered at 1 kHz and digitized at 20 kHz. Data acquisition and analysis were performed with pCLAMP9 software.
The inhibition of Ca 2ϩ 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 ␣ 2B -and ␣ 2C AR subtypes). The presence of prazosin had no significant effect on the inhibition of Ca 2ϩ currents by clonidine or guanfacine, confirming an ␣ 2A AR-mediated process (42). The peak inhibition of Ca 2ϩ currents by clonidine in the presence and absence of prazosin is 26.03 Ϯ 1.85 (n ϭ 15) and 30.07 Ϯ 2.45 (n ϭ 9), respectively, p ϭ 0.26. The peak inhibition of Ca 2ϩ currents by guanfacine in the presence and absence of prazosin is 24.42 Ϯ 2.16 (n ϭ 14) and 28.16 Ϯ 2.51 (n ϭ 9), respectively, p ϭ 0.31. All recordings were normalized to Ca 2ϩ rundown. To block receptor internalization, neurons were preincubated with ConA (160 g/ml) or PAO (10 M) for 20 min before recording and addition of ligands.
Intact Cell Receptor Phosphorylation-Intact cell phosphorylation of HA-␣ 2A AR in response to different agonists was performed as described previously (43) using HEK293 cells stably expressing murine HA-␣ 2A AR (31). Cells prelabeled with [ 32 P]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-␣ 2A AR protein). To examine the effect of ConA on ␣ 2A AR phosphorylation, cells were pretreated with 160 g/ml ConA before agonist stimulation.
Statistical Analysis-Statistical analyses were performed with the GraphPad Prism software using Student's t tests. p Ͻ 0.05 was considered statistically significant.

Generation and Characterization of HA-␣ 2A AR Knock-in
Mice-We utilized a homologous recombination gene targeting strategy to introduce the HA epitope-tagged wild type ␣ 2A AR 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 ␣ 2 AR 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-␣ 2A AR knock-in mouse line (Fig. 1, B and C).
Expression density of HA-␣ 2A AR in the knock-in mice, which is controlled by the endogenous ADRA2A locus, was indistinguishable from that of the ␣ 2A AR in WT mice, based on saturation binding analysis for the ␣ 2A AR in particulate preparations from the brains of WT or HA-␣ 2A AR knock-in mice (Fig. 1D). Similarly, affinity for the endogenous ligand, epineph-rine, was indistinguishable between the HA-␣ 2A AR in knock-in mice and ␣ 2A AR 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-␣ 2A AR to activate G proteins in response to epinephrine in native tissues, as demonstrated by epinephrine-stimulated [ 35 S]GTP␥S binding assays on brain particulate preparations from knock-in or WT mice (Fig. 1F). These data indicate that the knock-in HA-␣ 2A AR 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 ␣ 2A AR in WT mice. Therefore, the HA-␣ 2A AR knock-in mouse provides an elegant model to examine ␣ 2A AR trafficking and signaling in native cells expressing the endogenous receptor at its characteristic physiological density.
Distribution of Knock-in HA-␣ 2A AR in Mouse Brain and SCG-Using an HA antibody and a fluorescenceconjugated secondary antibody, we were able to examine the distribution of HA-␣ 2A AR 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 ␣ 2A AR 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-␣ 2A AR 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-␣ 2A AR (Fig. 2C) in the absence and presence of agonist stimulation is indistinguishable from that of the ␣ 2A AR obtained from WT mice (Fig. 2D). WT ␣ 2A AR was detected by an antibody directed against the C-terminal tail of the ␣ 2A AR (34). These data confirm that the knock-in HA-␣ 2A AR can be reliably used for examining ␣ 2A AR trafficking in native SCG neurons in parallel with our functional studies.
Clonidine and Guanfacine Differentially Promote ␣ 2A AR Internalization in Primary Cultured SCG Neurons-We examined surface HA-␣ 2A AR trafficking in response to stimulation by clonidine or guan- facine in cultured SCG neurons. Following 5 min of treatment with 10 Ϫ6 M clonidine or guanfacine, internalization of cell surface HA-␣ 2A AR 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-␣ 2A AR had moved from the surface into cytosolic compartments and formed puncta in a perinuclear area (Fig. 3A). In contrast, only scattered puncta of HA-␣ 2A AR were observed inside cells following incubation with guanfacine for 15 min (Fig. 3A). The extent of internalization of cell surface HA-␣ 2A AR in response to clonidine was about 3.1-fold greater than that induced by guanfacine (Fig.  3B). A similar phenomenon was observed with 10 Ϫ5 M of clonidine and guanfacine (Fig. 3B). These data provide the first documentation that clonidine and guanfacine differentially evoke the trafficking of the endogenous ␣ 2A AR in native sympathetic neurons.
Clonidine Induces Greater Phosphorylation of HA-␣ 2A AR 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-␣ 2A AR in response to clonidine compared with guanfacine, we examined whether clonidine and guanfacine exhibit different binding affinities or intrinsic activities at the ␣ 2A AR, under the same experimental settings. Fig. 4A demonstrates that the HA-␣ 2A AR has an indistinguishable affinity for clonidine and guanfacine, based on competition of these agents for [ 3 H]RX821002 binding. Binding studies with the nonhydrolyzable GTP analogue, [ 35 S]GTP␥S, indicate that these two agonists display a similar efficacy and potency in stimulating coupling of the occupied ␣ 2A AR to G proteins (Fig. 4B). These data suggest that different abilities for inducing ␣ 2A AR trafficking exhibited by clonidine and guanfacine cannot be attributed to receptor affinity or intrinsic activity of these two ligands at the ␣ 2A AR.
We have previously found that receptor phosphorylation is required for arrestin-mediated internalization of the ␣ 2A AR. 5 Therefore, we examined the relative ability of these two agents to evoke ␣ 2A AR phosphorylation using HEK293 cells stably expressing HA-␣ 2A AR. As shown in Fig. 4C, clonidine evokes a significantly higher level of ␣ 2A AR phosphorylation than guanfacine at the same concentration of ligand (10 Ϫ6 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 Ca 2ϩ 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 ␣ 2A AR phosphorylation and internalization might result in different desensitization profiles of ␣ 2A AR-mediated signaling processes in response to these two drugs. To test this hypothesis, we evaluated ␣ 2A AR-mediated inhibition of voltage-gated Ca 2ϩ 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 Ϫ5 M clonidine or guanfacine, the concentration that leads to maximum occupancy of the ␣ 2A AR (Fig. 4A), to evaluate the maximal desensitization rate of ␣ 2A AR-mediated inhibition of Ca 2ϩ currents by these two agonists. Clonidine and guanfacine treatment induced a similar level of acute inhibition (ϳ30%) of Ca 2ϩ currents (Fig. 5A). However, the sustained inhibitory effects of these two agents on Ca 2ϩ currents were remarkably different. Specifically, inhibition of Ca 2ϩ currents by clonidine began to wane after 200 s of incubation with this agonist, and dropped to ϳ25% of the maximal inhibition level after 5 min of incubation (Fig. 5B), whereas inhibition of Ca 2ϩ currents by guanfacine was maintained through the 5-min time point   (Fig. 5C). After 15 min of exposure to the agonist, clonidineevoked inhibition of Ca 2ϩ currents continued to drop to nearly 50% of the maximal inhibition level (Fig. 5B), whereas  only a slight decrease in Ca 2ϩ current inhibition was observed in response to guanfacine (Fig. 5C). These data indicate that clonidine induces a faster and more dramatic desensitization of ␣ 2A AR-evoked inhibition of Ca 2ϩ 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 ␣ 2A AR-mediated inhibition of Ca 2ϩ currents induced by clonidine as compared with guanfacine. Therefore, we evaluated the desensitization of Ca 2ϩ current inhibition evoked by 10 Ϫ5 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)(48)(49). Preincubation with ConA blocked HA-␣ 2A AR internalization in SCG neurons induced by either clonidine or guanfacine (Fig. 6A). More interestingly, ConA treatment attenuated desensitization of clonidine-induced inhibition of Ca 2ϩ 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 timedependent contribution of receptor internalization on desensitization of clonidine-induced ␣ 2A ARmediated inhibition of Ca 2ϩ currents. Although significant desensitization of guanfacineevoked inhibition of Ca 2ϩ currents is detected after 15 min of treatment, ConA blockade of ␣ 2A AR internalization had no effect on this desensitization (Fig. 6C), suggesting that guanfacine-induced desensitization does not require ␣ 2A AR internalization. Taken together, these data suggest that differential desensitization of the endogenous ␣ 2A AR in native sympathetic neurons to clonidine versus guanfacine relies on the ability of clonidine to rapidly and markedly promote ␣ 2A AR internalization.

HA-␣ 2A AR Knock-in Mouse Line Permits Assessment of Trafficking and Signaling Relationships in Native Target Cells-Our
data reveal that N-terminal HA-tagged ␣ 2A AR in a knock-in mouse fully mimics the WT receptor in terms of ␣ 2A AR 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 ␦-opioid receptor (54), have been shown to exhibit altered receptor densities. Furthermore, our knock-in HA-␣ 2A AR manifests an agonist-evoked redistribution profile that is indistinguishable from the WT ␣ 2A AR  (Fig. 2). Thus, the HA-␣ 2A AR 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-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 -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-␣ 2A AR knock-in mice, we demonstrated that clonidine induces a more dramatic internalization of the ␣ 2A AR 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 ␣ 2A AR phosphorylation than guanfacine (Fig.  4C), thus fostering an enhanced interaction of arrestin with the receptor (76), which leads to a more rapid and extensive ␣ 2A AR 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 ␤-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 postendocytosis, may contribute as much as 20 -30% to the desensitization process of ␤ 2 AR-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 ␣ 2A AR to achieve maximal desensitization of ␣ 2A AR-evoked inhibition of Ca 2ϩ 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 ␣ 2A AR-evoked Ca 2ϩ 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 ␣ 2A AR 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 voltagegated Ca 2ϩ 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 ␣ 2A AR-mediated inhibition of voltage-gated Ca 2ϩ channel currents than does guanfacine is likely of significance in vivo, because ␣ 2A AR-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 ␣ 2A AR, 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.