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J. Biol. Chem., Vol. 276, Issue 50, 47512-47517, December 14, 2001

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Activation and Deactivation Kinetics of _2A- and _2C-Adrenergic Receptor-activated G Protein-activated Inwardly Rectifying K⁺ Channel Currents^*

Moritz Bünemann, Markus M. Bücheler, Melanie Philipp, Martin J. Lohse, and Lutz Hein

From the Institut für Pharmakologie und Toxikologie, Universität Würzburg, 97078 Würzburg, Germany

Received for publication, September 7, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although G protein-coupled receptor-mediated signaling is one of the best studied biological events, little is known about the kinetics of these processes in intact cells. Experiments with neurons from _2A-adrenergic receptor knockout mice suggested that the _2A-receptor subtype inhibits neurotransmitter release with higher speed and at higher action potential frequencies than the _2C-adrenergic receptor. Here we investigated whether these functional differences between presynaptic ₂-adrenergic receptor subtypes are the result of distinct signal transduction kinetics of these two receptors and their coupling to G proteins. _2A- and _2C-receptors were stably expressed in HEK293 cells at moderate (~2 pmol/mg) or high (17-24 pmol/mg) levels. Activation of G protein-activated inwardly rectifying K⁺ (GIRK) channels was similar in extent and kinetics for _2A- and _2C-receptors at both expression levels. However, the two receptors differed significantly in their deactivation kinetics after removal of the agonist norepinephrine. _2C-Receptor-activated GIRK currents returned much more slowly to base line than did _2A-stimulated currents. This observation correlated with a higher affinity of norepinephrine at the murine _2C- than at the _2A-receptor subtype and may explain why _2C-adrenergic receptors are especially suited to control sympathetic neurotransmission at low action potential frequencies in contrast to the _2A-receptor subtype.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

G protein-coupled receptors (GPCRs)¹ transfer a large diversity of extracellular signals into the cell interior, including light, neurotransmitters, and hormones. Although GPCRs represent some of the best studied signaling molecules, relatively little information exists about the kinetics of signal transduction by these receptors (except for rhodopsin) in intact cells. However, more detailed knowledge about the kinetic properties of GPCR signal transduction would be of particular interest to determine the physiological significance of closely related receptor subtypes, which can be activated by the same endogenous agonist but differ in their biological function.

Functional data on ₂-adrenergic receptor subtypes suggest that they differ in their signaling kinetics. Interestingly, several physiological differences were identified between presynaptic _2A- and _2C-receptor subtypes (1). In mouse atria, the _2A-subtype inhibited norepinephrine release at high stimulation frequencies whereas the _2C-receptor operated at lower levels of sympathetic nerve activity (1). Moreover, inhibition of norepinephrine release mediated by the _2A-subtype occurred much faster than inhibition by the _2C-receptor. These findings indicate that two presynaptic receptors in the inhibitory feedback loop of transmitter release may differentially regulate synaptic transmission. Several explanations may account for these functional differences. ₂-Adrenergic receptor subtypes have been shown to differ in their signal transduction, agonist-dependent internalization and receptor trafficking, subcellular localization, and binding to intracellular scaffolding proteins (2-11).

₂-Adrenergic receptors are essential regulators of the sympathetic and central nervous system (12-15). Three subtypes of ₂-adrenergic receptors have been identified in different species (12). Recently, transgenic mouse models lacking individual ₂-receptor subtypes have been generated to define the physiological significance and therapeutic potential of these receptor subtypes (for reviews, see Refs. 16-18). ₂-Receptors are essential constituents of a negative feedback loop regulating presynaptic neurotransmitter release (15). Experiments with gene-targeted mice lacking individual ₂-adrenergic receptors have demonstrated that two ₂-receptor subtypes, _2A and _2C, serve as presynaptic regulators in the sympathetic nervous system and in the central nervous system (1, 19). Mice lacking both of these receptors had enhanced circulating norepinephrine levels and developed cardiac hypertrophy and failure (1).

We therefore sought a simple experimental model to investigate potential differences in the speed of _2A- and _2C-receptor-mediated signaling and the role of receptor levels for signaling kinetics. We have determined the properties of these receptor subtypes to inhibit N-type Ca²⁺ channels or activate G protein-activated inwardly rectifying K⁺ (GIRK) channels in a heterologous expression system in human embryonic kidney (HEK293) cells. We found that, at similar levels of expression, _2A- and _2C-receptors did not differ in their activation kinetics, but the deactivation after receptor stimulation by norepinephrine was significantly slower for _2C- than for _2A-receptors. This finding is consistent with a higher affinity of norepinephrine for _2C- than for _2A-receptors. Different deactivation kinetics may account for part of the functional differences between presynaptic ₂-adrenergic receptor subtypes to enhance the power of synaptic regulation and plasticity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

₂-Receptor-expressing Cell Lines and Transfection Procedure-- HEK293 cells were stably transfected with the murine _2A- and _2C-adrenergic receptors using a pcDNA3 vector (2). After selection with 200 µg/ml G418 (Life Technologies, Inc.), stable cell clones were screened by radioligand binding and homogeneity of receptor expression was controlled by immunofluorescence (2). _2A-AR-or _2C-AR-expressing cell lines were transiently transfected with a bicistronic expression vector encoding for GIRK1 and GIRK4 (kindly provided by Dr. L.Y. Jan, Howard Hughes Medical Institute, University of California, San Francisco, CA) as well as with H3 plasmids encoding the CD8 receptors (0.15 µg/5-cm dish; kindly provided by Dr. G Yellen, Harvard University, Cambridge, MA) using the Effectene transfection kit (Qiagen, Hilden, Germany). For GIRK deactivation studies, either rat RGS4 (in pcDNA3.1) or empty pcDNA3 vector (0.5 µg/5-cm dish) was added to the transfection reaction. 18-24 h after transfection, cells were replated onto poly-L-lysine-coated cell culture dishes and were used for experiments 40-50 h after transfection. Successfully transfected cells were detected by binding Dynabeads coated with anti-CD8 antibodies (Dynal ASA, Oslo, Norway) to the cells (20). For measurement of N-type Ca²⁺ channels, G1A1 cells (Ref. 21; cells provided by Dr. R. J. Miller with permission from SIBIA Neuroscience, La Jolla, CA) were transiently transfected with pCDNA3-_2A or pcDNA3-_2C (1 µg/5-cm dish) and H3-CD8 (0.15 µg/5-cm dish) using Effectene.

Immunofluorescence Staining-- Immunofluorescence detection of ₂-adrenergic receptor was performed as described previously (2) using _2A- and _2C-adrenergic receptor subtype-specific antibodies (2, 22) and secondary anti-mouse Alexa (Molecular Probes, Leiden, Netherlands) and anti-mouse Cy2 antibodies (Dianova, Hamburg, Germany).

Radioligand Binding-- Expression levels of ₂-adrenergic receptors were determined after lysis of cells with hypotonic buffer (5 mM Tris, 2 mM EDTA, pH 7.4) as described (23). [³H]RX 821002 (Amersham Pharmacia Biotech, Freiburg, Germany) was used as radioligand, and nonspecific binding was determined by addition of 1 µM atipamezole (Orion Corp., Turku, Finland). For competition assays, 4 nM [³H]RX 821002 was used with equal amounts of _2A- and _2C-receptors (150 fmol) (24).

Electrophysiology-- Inward currents through GIRK channels were measured by whole cell patch recording similar to that described previously (25). Briefly, whole cell membrane currents were measured at 90 mV holding potential and agonist-induced currents were activated by fast superfusion of the cells using a solenoid valve operated superfusion system (26). Solution exchange occurred within less than 150 ms. Voltage ramps (from 120 mV to +60 mV in 500 ms, every 10 s) were used to determine current-voltage relationships. Barium currents through N-type Ca²⁺ channels were measured as described (27). Depolarization-evoked Ba²⁺ currents were measured every 10 s using 25-ms test pulses of +10 mV (holding potential was 90 mV). All measurements were performed at room temperature.

Patch pipettes were fabricated from borosilicate glass capillaries (GF-150-10, Harvard Apparatus, Edenbridge, United Kingdom) using a horizontal puller (P-95, Sutter Instruments, Novato, CA). The DC resistance of the filled pipettes ranged from 2 to 5 megohms. Membrane currents were recorded using either an EPC 9 patch clamp amplifier (HEKA, Lambrecht, Germany) or an Axopatch 200 (Axon Instruments, Union City, CA). Signals were analog-filtered using a low pass Bessel filter (1-3-kHz corner frequency). Data were digitally stored using computers equipped with the appropriate hardware/software package (ISO2 by MFK, Frankfurt/Main, Germany; or PULSE by HEKA, Lambrecht, Germany) for voltage control, data acquisition, and data evaluation.

For the measurement of GIRK currents, an extracellular solution of the following composition was used: 120 mM NaCl, 20 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES-NaOH (pH 7.3). The internal (pipette) solution contained: 100 mM potassium aspartate, 40 mM KCl, 5 mM MgATP, 10 mM HEPES-KOH, 5 mM NaCl, 2 mM EGTA, 2 mM MgCl₂, 0.01 mM GTP (pH 7.3). For measurement of barium currents through N-type Ca²⁺ channels, the extracellular solution contained: 105 mM NaCl, 25 mM CsCl, 10 mM BaCl₂, 1 mM MgCl₂, 10 mM HEPES-NaOH (pH 7.3). The intracellular solution contained 130 mM CsCl, 5 mM EGTA, 5 mM HEPES-CsOH, 5 mM MgATP, 1 mM MgCl₂, 0.01 mM GTP (pH 7.3). NaCl, KCl and MgCl₂ were purchased from Merck (Darmstadt, Germany); potassium aspartate, CaCl₂, BaCl₂, CsCl, ATP, GTP, EGTA, norepinephrine, and phenylephrine were from Sigma (Deisenhofen, Germany); and HEPES was from Biomol (Hamburg, Germany).

Statistics-- Results shown display means ± S.E. for n = 5-10 experiments if not stated otherwise, and statistically significant differences were determined by applying the Student's t test for two independent data sets (*, p  0.05; **, p  0.01).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ca²⁺ Channel Inhibition and GIRK Activation by ₂-Adrenergic Receptors-- The ability of ₂-adrenergic receptor subtypes _2A and _2C to activate GIRK currents or to inhibit N-type Ca²⁺ currents was initially tested in HEK293 cells transiently expressing either one of the receptor subtypes and GIRK channels or in HEK cells stably expressing N-type Ca²⁺ channels composed of _1B-, _1B-, and ₂-channel subunits (G1A1 cells; Ref. 21). Activation of both adrenergic receptors, _2A and _2C, led to inhibition of N-type Ca²⁺ currents (Fig. 1) and to activation of GIRK channels (Fig. 1). The time course of the phenylephrine-induced inhibition of Ba²⁺ currents through N-type Ca²⁺ channels was similar for the two ₂-receptor subtypes (data not shown). In addition, the time course of the activation of GIRK currents was indistinguishable from the time course of the phenylephrine-induced inhibition of N-type Ca²⁺ channels (Fig. 1). However, independent from the effector system, the time courses of ₂-adrenergic responses were quite variable in this transient expression system, possibly because of cell to cell variability of the expression levels of the receptors. Therefore, we chose to investigate the signaling of both receptor subtypes in stable HEK293 cell lines expressing defined amounts of receptor. From a total of 48 cell lines with ~0.1-50 pmol of receptor/mg of membrane protein, four cell lines were chosen for further experiments (Fig. 2). An intermediate level (2 pmol of receptor/mg of membrane protein) and a high level (17-24 pmol of receptor/mg of membrane protein) of receptor expression were used for all experiments. Lower expression levels of ₂-receptors resulted in less reproducible Ca²⁺ channel inhibition or GIRK channel activation, respectively (data not shown). As previous studies had indicated that _2A- and _2C-receptors may differ in their plasma membrane targeting (2, 11), all cell clones used for this study were screened by immunofluorescence staining and only clones with plasma membrane localization of ₂-receptors were included for further study (Fig. 2, c and d). As the GIRK channel activation kinetics by ₂-receptors had been found to be similar to the kinetics of ₂-receptor-mediated inhibition of N-type Ca²⁺ channels, and as measurement of GIRK current activation allowed for a higher temporal resolution of the ₂-response, we focused on these channels for detailed kinetic analysis of ₂-receptor signaling.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Comparison of 2C-receptor-induced responses on N-type Ca2+ currents and GIRK currents. HEK293 cells or HEK G1A1 cells stably expressing 1B, 1B, and 2 subunits of N-type Ca2+ channels were transiently transfected with 2C-adrenergic receptors and GIRK1 and GIRK4 channel subunits (only HEK293 cells) and were subjected to whole cell patch experiments. The time course of the inhibition of N-type Ca2+ channels (black squares) as well as the activation of GIRK currents (representative current trace) by the -agonist phenylephrine were determined. Ba2+ currents through N-type Ca2+ channels were elicited every 10 s by a depolarizing voltage pulse from 90 mV to +10 mV for 25 ms. Application of the agonist 1, 2, 3, 4, 5, and 10 s prior to a voltage pulse allowed us to follow the time course of the current inhibition. For comparison both 2C-receptor-mediated responses were normalized to the maximal response.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Expression of 2A- and 2C-adrenergic receptors in HEK293 cells. Cells were stably transfected with murine 2A- or 2C-receptors, and clones with similar expression levels (a and b) and cell surface expression of receptors (c and d) were selected for this study. a and b, saturation binding experiments for [3H]RX821002 to 2A- and 2C-receptors. Nonspecific binding was determined in the presence of 1 µM atipamezole. c and d, subcellular distribution of 2-adrenergic receptors was detected by immunofluorescence staining in permeabilized HEK293 cells stably expressing high levels of 2A- or 2C-receptors. Bars, 10 µm.

₂-Receptor-mediated Activation of GIRK Currents-- Activation of ₂-receptors by norepinephrine led to a concentration-dependent increase in steady-state GIRK currents (Fig. 3). In cells expressing similar levels of receptors, the concentration-response curves were indistinguishable for the _2A- and the _2C-receptor. For both receptor subtypes, the potency for norepinephrine was closely correlated with the density of receptor expression. A 10-fold increase in receptor expression led to a similar increase in the potency of norepinephrine to activate steady-state GIRK currents. To test whether the kinetics of ₂-receptor activation differed between the two ₂-receptor subtypes, the time course of GIRK current activation by norepinephrine was determined (Fig. 4). With increasing concentrations of norepinephrine (10 nM to 10 µM), the time for half-maximal GIRK activation decreased progressively. At 2 pmol/mg of ₂-receptor expression, the half-times for GIRK activation were similar for _2A- and _2C-receptors at all three norepinephrine concentrations tested. Only at high receptor levels and at 10 µM norepinephrine, concentrations 10⁴ times the EC₅₀, the activation of GIRK currents was significantly faster when activated via _2A-receptors (218 ± 13 ms) as compared with _2C-receptors (503 ± 29 ms, p < 0.05) (Fig. 4).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Concentration-response curves of norepinephrine-induced GIRK current activation by 2A- and 2C-adrenergic receptors in HEK293 cells. The amplitudes of norepinephrine-induced GIRK currents (steady-state currents) were measured in cell clones expressing high (2A, 24 pmol/mg; 2C, 17 pmol/mg) or intermediate (2A and 2C, 2 pmol/mg) levels of 2-adrenergic receptors. At similar levels of expression, concentration-response curves were essentially identical for both 2-receptor subtypes. However, for both receptor subtypes, norepinephrine concentration-response curves were shifted to the left in cells expressing high amounts of 2-receptors compared with 10-fold lower receptor levels. Data shown represent means ± S.E. for 7-10 experiments performed in two to three different transfections.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Activation kinetics of GIRK currents induced by norepinephrine. The activation of inward GIRK currents in response to fast superfusion with norepinephrine (using different concentrations) was recorded. Representative current recordings (left side, normalized to maximal responses) and summarized data for the time of half-maximal activation (right) are shown for cell lines expressing indicated levels of 2A- or 2C-receptors. Data shown represent means ± S.E. of 5-10 different cells derived from two to three different transfections.

The activation kinetics of norepinephrine-induced GIRK currents were not only dependent on the agonist concentration, but also correlated with the receptor expression level independent of the receptor subtype, e.g. the half-time for GIRK current activation by 10 nM norepinephrine decreased from 5.3 ± 1.1 s to 2.1 ± 0.3 s when _2A-receptor expression was increased from 2 pmol/mg to 17 pmol/mg (similar results were obtained for the _2C-receptor, Fig. 4). Thus, with the possible exception of maximal stimulation of high levels of ₂-receptors, no subtype-specific differences could be identified for activation kinetics or steady-state GIRK currents. However, both parameters were greatly dependent on the level of receptor expression for the two ₂-receptor subtypes.

Deactivation Kinetics of GIRK Currents-- To search for subtype-specific differences in off-rates of ₂-receptor-activated GIRK transients, the time course of deactivation of GIRK currents was monitored after rapid removal of norepinephrine (Fig. 5). For both ₂-receptors, GIRK currents completely returned to base-line values within 60 s. However, a significant difference remained between the deactivation time courses of the two receptor subtypes; _2A-stimulated GIRK currents deactivated 2-3.5 times faster than _2C-stimulated GIRK currents (Fig. 5, b and c). This difference was the same at high receptor levels (Fig. 5, a and b) and at moderate levels (Fig. 5c). For both receptor subtypes, the half-time for deactivation was similar at moderate and at high levels of receptor expression.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Deactivation kinetics of 2-adrenergic receptor-activated GIRK currents after removal of norepinephrine. GIRK currents were first activated via 2A- or 2C-receptors by superfusing the cells with norepinephrine until reaching a steady state, and the time course of GIRK current deactivation was then measured in response to rapid washout of norepinephrine (a, representative recordings). The times until reaching half-maximal deactivation were summarized (b) and were expressed as means ± S.E. (n = 5-8). c, norepinephrine displaced the 2-receptor antagonist, [3H]RX821002, at lower concentrations from 2C- than from 2A-receptors (Ki for 2A, 5.0 µM; for 2C, 0.8 µM).

To test whether different receptor affinities for norepinephrine are responsible for the observed deactivation kinetics, competition binding experiments were performed with HEK cells expressing _2A- and _2C-receptors. The K_i values for norepinephrine were higher at the _2A-receptor (5.03 µM) than at the _2C-subtype (0.77 µM), indicating that, indeed, norepinephrine has a higher affinity for the _2C-receptor than for the _2A-receptor (Fig. 5d).

The higher affinity of norepinephrine for the _2C-receptor compared with the _2A-receptor suggested a slower time constant for the unbinding of the agonist from the _2C-receptor. We hypothesized this phenomenon to be responsible for the slower deactivation kinetics of the _2C-receptor-mediated responses. To rule out that the slower deactivation of the _2C-adrenergic receptor was caused by a post-receptor mechanism, the regulator of G protein-signaling protein, RGS4, was transiently overexpressed in these cells (Fig. 6). RGS4 has been shown to accelerate deactivation of G proteins 100-1000-fold (28) and to substantially accelerate GIRK current deactivation after agonist removal (29, 30). If indeed the slower deactivation of _2C-receptors were the result of decreased G protein inactivation, RGS4 overexpression should lead to enhanced turn-off rates of the G protein. However, addition of RGS4 did not affect the deactivation of ₂-receptor-activated GIRK currents (Fig. 6). Interestingly, phenylephrine, an -adrenergic receptor agonist with very low affinity, showed significantly faster deactivation of GIRK currents than norepinephrine, when used at equally potent concentrations (Fig. 6). These findings indicate that the agonist affinity rather than intrinsic receptor-G protein interaction determined the turning off rates of the ₂-receptor evoked responses.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Receptor rather than G protein deactivation limits deactivation kinetics of 2C-adrenergic receptor-activated GIRK currents after removal of norepinephrine. GIRK currents in cells expressing 2C-adrenergic receptor at 2 pmol/mg protein were activated with either 10 nM norepinephrine or 1 µM phenylephrine, and deactivation kinetics were determined (see representative current recording in a and summarized data shown in b; n = 7-8). In addition, the effect of coexpression of RGS4 on the deactivation kinetics of currents activated by 10 nM norepinephrine was analyzed (n = 4).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our study represents the first in which activation and deactivation of G_i protein signaling in intact cells were determined in dependence of defined amounts of receptor expression. No subtype-dependent differences were found in steady-state GIRK currents and their activation kinetics between _2A- and _2C-receptors expressed at moderate or at high levels in HEK293 cells. However, for both receptor subtypes, a 10-fold increase in receptor expression was correlated with a 2.5-3-fold acceleration of the GIRK current activation for both receptor subtypes.

Most interestingly, _2A- and _2C-adrenergic receptors differed significantly in their deactivation kinetics. This finding may explain part of the functional differences between the two ₂-receptor subtypes, which have been identified in mouse sympathetic neurons in vitro (1). At high stimulation frequencies of isolated mouse atria, norepinephrine release was primarily controlled by the _2A-receptor subtype, whereas at lower frequencies the _2C-receptor was the principle regulator of transmitter release (1).

Deactivation of norepinephrine-activated GIRK currents was significantly faster for the _2A- than for the _2C-receptor. This finding is consistent with the higher affinity (and thus slower dissociation kinetics) of norepinephrine for the _2C-subtype than for the _2A-receptor subtype (31). Deactivation of the _2C-receptor-induced GIRK currents occurred with faster kinetics, when the low affinity agonist phenylephrine was used instead of the endogenous agonist, norepinephrine. In addition, enhancing the rate of G protein inactivation by overexpressing RGS4 in these cells did not alter GIRK deactivation. Taken together, these results indicate that agonist dissociation rather than receptor-G protein interaction or a post-receptor effect was responsible for the slower turn-off rate of the norepinephrine-activated _2C-receptor.

The finding of similar activation kinetics is in contrast to in vitro observations in intact sympathetic neurons where endogenous _2A-receptors can inhibit presynaptic transmitter release significantly faster than _2C-receptors (1). Several possibilities remain to explain this discrepancy between the HEK293 cell data of this study and the situation in intact neurons. Expression levels may differ between ₂-receptor subtypes. Indeed, in the central nervous system, ~90% of ₂-adrenergic receptors belong to the _2A-subtype, whereas only 10% are _2C-receptors (32). The _2B-receptor is restricted to very few brain nuclei and thus does not contribute significantly to total brain ₂-receptor binding. However, these data are based on radioligand binding experiments in membrane homogenates, and no data are available on receptor densities in presynaptic membranes.

Selective targeting of ₂-receptor subtypes to specific membrane domains has been shown in a number of different cell types (2, 9, 11). Whereas _2A-receptors were always targeted to the plasma membrane, _2C-receptors were found in addition in an intracellular membrane compartment (22). Specific targeting of _2A- and _2C-receptors to distinct plasma membrane domains in intact neurons may account for differences in signaling kinetics because of their distance from the neurotransmitter release site or because of specific interaction with distinct intracellular messenger systems. Different modulatory pathways have been implicated in presynaptic inhibition of neurotransmission by G protein-coupled receptors (33), which also differ in their signaling kinetics. In sympathetic neurons norepinephrine, via G_o, slowed the kinetics of Ca²⁺ current activation and a second, sustained reduction in Ca²⁺ current, via the inhibitory G protein, G_i, was also observed (5). Thus further studies are required to test whether indeed _2A- and _2C-receptors in intact neurons in vivo differ in their subcellular localization and/or intracellular signaling pathways. These studies demonstrate that subtype-specific differences in receptor signaling kinetics may be important avenues for future study to further understand the functional significance of GPCR subtype diversity.

	ACKNOWLEDGEMENTS

We are indebted to Kerstin Hadamek for assistance with immunofluorescence screening of stable HEK cell clones. We thank M. Marlene Hosey and R. ten Eick (Northwestern University, Chicago, IL) for fruitful discussions and for providing equipment and laboratory space.

	FOOTNOTES

^* This work was supported by Grant SFB487 and a Leibniz award from the Deutsche Forschungsgemeinschaft, by the Fond der Chemischen Industrie, and by a grant from the Boehringer Ingelheim Foundation (to M. M. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Inst. für Pharmakologie und Toxikologie, Universität Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany. Tel.: 49-931-201-5401; Fax: 49-931-201-3539; E-mail: hein@toxi.uni-wuerzburg.de.

Published, JBC Papers in Press, October 8, 2001, DOI 10.1074/jbc.M108652200

	ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; ₂-AR, ₂-adrenergic receptor; GIRK, G protein-activated inwardly rectifying K⁺ channel; HEK, human embryonic kidney.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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