Differential response of Drosophila cell lines to extracellular adenosine

Adenosine (Ado) is a crucial metabolite that affects a wide range of physiological processes. Key proteins regulating Ado signaling, transport and metabolism are conserved among vertebrates and invertebrates. It is well known that Ado influences proliferation of several vertebrate and invertebrate cells. Here we show that Ado negatively influences viability, changes morphology and mitochondrial polarity of the Drosophila imaginal disc cell line (Cl.8+) via a mechanism exclusively dependent on cellular Ado uptake. High transport of Ado is followed by phosphorylation and ATP production as a part of Ado salvation, which at higher concentrations may interfere with cellular homeostasis. In contrast, hematopoietic cell line Mbn2, which grows well in high Ado concentration, preferentially uses adenosine deaminase as a part of the purine catabolic pathway. Our results show that different types of Drosophila cell lines use different pathways for Ado conversion and suggest that such differences may be an important part of complex mechanisms maintaining energy homeostasis in the body.     

Identification of four Drosophila allatostatins as the cognate ligands for the Drosophila orphan receptor DAR-2

The allatostatins are generally inhibitory insect neuropeptides. The Drosophila orphan receptor DAR-2 is a G-protein-coupled receptor, having 47% amino acid residue identity with another Drosophila receptor, DAR-1 (which is also called dros. GPCR, or DGR) that was previously shown to be the receptor for an intrinsic Drosophila A-type (cockroach-type) allatostatin. Here, we have permanently expressed DAR-2 in CHO cells and found that it is the cognate receptor for four Drosophila A-type allatostatins, the drostatins-A1 to -A4. Of all the drostatins, drostatin-A4 (Thr-Thr-Arg-Pro-Gln-Pro-Phe-Asn-Phe-Gly-Leu-NH(2)) is the most effective in causing a second messenger cascade (measured as bioluminescence; threshold, 10(-9) M; EC(50), 10(-8) M), whereas the others are less effective and about equally potent (EC(50), 8 x 10(-8) M). Northern blots showed that the DAR-2 gene is expressed in embryos, larvae, pupae, and adult flies. In adult flies, the receptor is more strongly expressed in the thorax/abdomen than in the head parts, suggesting that DAR-2 is a gut receptor. This is confirmed by Northern blots from 3rd instar larvae, showing that the DAR-2 gene is mainly expressed in the gut and only very weakly in the brain. The Drosophila larval gut also contains about 20-30 endocrine cells, expressing the gene for the drostatins-A1 to -A4. We suggest, therefore, that DAR-2 mediates an allatostatin (drostatin)-induced inhibition of gut motility. This is the first report on the permanent and functional expression of a Drosophila gut neurohormone receptor.     

Two distinct pathways for cAMP-mediated down-regulation of the beta 2-adrenergic receptor. Phosphorylation of the receptor and regulation of its mRNA level

We have studied cyclic AMP-mediated regulation of the beta 2-adrenergic receptor (beta 2AR). The effects of cAMP were assessed in Chinese hamster fibroblast (CHW) cells expressing either the wild type human beta 2AR receptor (CH-beta 2) or mutated forms of the receptor lacking the consensus sequences for phosphorylation by the cAMP-dependent protein kinase. Treatment of the CH-beta 2 cells with the cAMP analogue dibutyryl cAMP (Bt2cAMP) induces a time-dependent "down-regulation" of the number of beta 2AR. This down-regulation of the receptors is accompanied by a decline in the steady state level of beta 2AR mRNA. Moreover, the treatment with Bt2cAMP induces an increase in the phosphorylation level of the membrane-associated beta 2AR. Both the reduction in beta 2AR mRNA and the enhanced phosphorylation of the receptor are rapid and precede the loss of receptor. The down-regulation of beta 2AR induced by Bt2cAMP is concentration-dependent and mimicked by the other biologically active cyclic nucleotide analogue, 8-Br-cAMP, by forskolin, and by the phosphodiesterase inhibitor, isobutylmethylxanthine. In the CHW cell lines expressing receptors lacking the putative protein kinase A phosphorylation sites, the Bt2cAMP-induced phosphorylation of beta 2AR is completely abolished. In these cells the down-regulation of beta 2AR receptor number produced by cAMP is significantly slowed, whereas the reduction in beta 2AR mRNA level is equivalent to that observed in CH-beta 2 cells. These data indicate that there are at least two pathways by which cAMP may decrease the number of beta 2ARs in cells: one involves phosphorylation of the receptor by the cAMP-dependent protein kinase and the other leads to a reduction in steady state beta 2AR mRNA levels.     

A role for the A3 adenosine receptor in determining tissue levels of cAMP and blood pressure: studies in knock-out mice

Adenosine administration has been reported to lower blood pressure by activating specific membrane receptors. The rat and human heart and aorta have been previously found to express both A2-type adenosine receptors, which activate adenylyl cyclase, and A3 adenosine receptors (A3AR), which inhibit adenylyl cyclase. In the current study, we used A3 adenosine receptor (A3AR) knock-out mice to examine the hypothesis that the relative levels of the A2-type adenosine receptors and A3AR determine the steady-state levels of cAMP in the cells and may affect blood pressure. We found that the A3AR knock-out mice express normal levels of the A1- and A2-type adenosine receptors. In situ hybridization demonstrated that the level of A3AR is high in the vascular smooth muscle layer of aortas derived from wild-type mice, but is not detectable in the knock-out mice. The steady-state level of cAMP is elevated in the aorta and heart of knock-out mice, as compared to wild-type mice, but is not altered in platelets, where A3AR is not expressed naturally. A3AR knock-out mice possess a blood pressure comparable to this in wild-type mice. However, when challenged with adenosine, the knock-out mice display a further increase in cAMP levels in the heart and vascular smooth muscle and a significant decrease in blood pressure, as compared to wild-type mice. In contrast, the effect of adenosine on ADP-induced platelet aggregation is similar in both types of mice. These studies indicate that the A3AR affects the steady-state level of cAMP in the tissues where it is naturally expressed, and that it influences the blood pressure in response to adenosine.     

Modulation of the cardiac pacemaker of Drosophila: cellular mechanisms

The myogenic cardiac pacemaker of Drosophila melanogaster responds to a range of neurotransmitters and hormones by adjusting heart rate. These cardioactive substances ultimately affect the activity of ion channels comprising the pacemaker. We report here work utilizing genetic variants and pharmacological tools to explore a subset of possible mechanisms for this cellular signaling, specifically: receptors, cAMP, cGMP, G proteins, and calcium. We found that alpha(1) adrenergic and 5-hydroxytryptamine(2) (5-HT(2)) receptors are critical components of mediating modulation of heart rate. There was no evidence that the cAMP system is part of the modulatory mechanism. cGMP is likely to be integral to one active pathway, as non-hydrolyzable forms of this cyclic nucleotide increase heart rate, and flies bearing the mutation sitter, a recessive allele of the foraging gene, which encodes a cGMP-dependent kinase, have tachycardia. Heart rhythm is affected by pertussis toxin and by agonists and antagonists of both alpha(1) adrenergic and 5-HT(2) receptors; this suggests involvement of two different types of G proteins. The l(4)16/ciD line, containing a mutation in CaM kinase II, eliminates pacemaker responsiveness to serotonin but is without effect on norepinephrine sensitivity. This result is the same as that for the CaM kinase II enzyme inhibitor KN-93. This work establishes a framework for further investigations into the control of the cardiac pacemaker, and expands the applicability of the Drosophila heart model.     

Influences of adenosine receptor antagonism on vasodilator responses to adenosine and exercise in adenosine responders and nonresponders.

We previously demonstrated a bimodal distribution of vasodilator responsiveness to adenosine (Ado) infusion in human subjects, despite similar responses to exercise between subgroups [subjects responsive to Ado infusion (Ado responders) and subjects with blunted vasodilator responses to Ado infusion (Ado nonresponders]). (Martin EA, Nicholson WT, Eisenach JH, Charkoudian N, and Joyner MJ. J Appl Physiol 101: 492-499, 2006). A component of this difference was attributed to a larger nitric oxide component of Ado-mediated vasodilation in responders. However, there may also be differences in Ado receptors between these subgroups. We hypothesized that Ado receptor antagonism would reduce vasodilator responsiveness to Ado and exercise only in Ado responders. To test this hypothesis, we compared forearm vasodilation induced by intra-arterial infusion of three doses of Ado to vasodilation during three workloads of forearm handgrip exercise before and after Ado receptor antagonism with aminophylline (Aph) in 19 subjects. In Ado responders, the change in forearm vascular conductance above baseline for the low, medium, and high doses of Ado, respectively, was 93 +/- 16, 140 +/- 14, 194 +/- 18 before Aph and 27 +/- 12, 71 +/- 19, and 134 +/- 34 ml.min(-1).100 mmHg(-1) after Aph (P < 0.05 for low and medium dose before vs. after Aph). For nonresponders, these values were 30 +/- 5, 39 +/- 6, and 78 +/- 9 ml.min(-1).100 mmHg(-1) before Aph (P < 0.05 vs. responders), with no difference after Aph (P > 0.05). We found that Ado receptor blockade significantly inhibited exercise hyperemia only at high workloads in both responders and nonresponders (P < 0.05 before vs. after Aph). We conclude that there may be reduced Ado receptor responsiveness or sensitivity in nonresponders. Furthermore, Ado may play a limited role exercise hyperemia in both subgroups.     

Stimulation of the proximal tubule Na+-ATPase activity by adenosine A(2A) receptor

The aim of this work was to determine the molecular mechanism involved in the stimulation of the pig kidney proximal tubule Na+-ATPase by adenosine (Ado). To study the role of A2 Ado receptors, we added in all experiments 10(-6)M DPCPX, an A1 receptor-selective antagonist, since we have previously shown that Ado inhibits the enzyme activity through this receptor. Ado increased the Na+-ATPase activity with maximal effect observed at 10(-6)M. The presence of both A(2A) and A(2B) receptors were demonstrated by immunoblotting using specific polyclonal antibodies. The stimulatory effect of Ado was completely abolished by 5 x 10(-9)M DMPX, an antagonist of A2 receptor, and 10(-7)M SCH 58261, an A(2A) receptor-selective antagonist. DMPA (10(-7)M), a specific agonist of A(2A) receptor mimicked the stimulatory effect of Ado. Involvement of a Gs protein/adenylate cyclase/PKA pathway was evidenced by: (a) the reversion of Ado-induced effect by GDPbetaS; (b) stimulation of the Na+-ATPase activity in a similar and non-additive manner to Ado by 10(-8)M cholera toxin, 10(-7)M GTPgammaS, 10(-6)M forskolin, 10(-7)M cAMP or 1.25 U catalytic subunit of PKA; (c) the reversion of the stimulatory effect of Ado by 10(-8)M PKA inhibitor peptide; (d) Ado-produced two-fold increase of the PKA activity, which was completely reversed by 10(-6)M DMPX. These are the first evidences showing the modulation of a renal primary active sodium transporter by Ado through A(2A) receptor.     

Characterization of the short neuropeptide F receptor from Drosophila melanogaster

A seven transmembrane G-protein coupled receptor has been cloned from Drosophila melanogaster. This receptor shows structural similarities to vertebrate Neuropeptide Y(2) receptors and is activated by endogenous Drosophila peptides, recently designated as short neuropeptide Fs (sNPFs). sNPFs have so far been found in neuroendocrine tissues of four other insect species and of the horseshoe crab. In locusts, they accelerate ovarian maturation, and in mosquitoes, they inhibit host-seeking behavior. Expression analysis by RT-PCR shows that the sNPF receptor (Drm-sNPF-R) is present in several tissues (brain, gut, Malpighian tubules and fat body) from Drosophila larvae as well as in ovaries of adult females. All 4 Drosophila sNPFs clearly elicited a calcium response in receptor expressing mammalian Chinese hamster ovary cells. The response is dose-dependent and appeared to be very specific. The short NPF receptor was not activated by any of the other tested arthropod peptides, not even by FMRFamide-related peptides (also ending in RFamide), indicating that the Arg residue at position 4 from the amidated C-terminus appears to be crucial for the response elicited by the sNPFs.     

Gene silencing of selected calcium-signalling molecules in a Drosophila cell line using double-stranded RNA interference

Using the Drosophila melanogaster S2 cell line, stably expressing a cloned muscarinic acetylcholine receptor (AChR), DM1, we have applied gene silencing by double-stranded RNA interference (RNAi) to knock down gene products involved in DM1-mediated calcium signalling. We have shown that RNAi knock down of either the inositol 1,4,5-trisphosphate receptor (Ins(1,4,5)P(3)R), or the SERCA calcium pump in the S2-DM1 cells blocks the increase in intracellular calcium concentration ([Ca(2+)](i)) resulting from activation of the DM1 receptor by 100 microM carbamylcholine (CCh). When RNAi designed to knock down the ryanodine receptor (RyR) was tested, there was no change in the calcium increase detected in response to CCh, consistent with a failure to detect RyRs in S2-DM1 cells using RT-PCR. A combination of RNAi and calcium imaging has provided a direct demonstration of key roles for the Ins(1,4,5)P(3)R and the SERCA pump in the response to DM1 receptor activation.Thus, we show that silencing of individual genes by RNAi in a well characterised Drosophila S2 cell line offers experimental opportunities for cell-signalling studies. Future investigations with RNAi libraries taking full advantage of the wealth of new information available from sequencing the Drosophila genome, may help identify novel components of cell-signalling pathways and functionally linked gene products.     

Molecular response of Drosophila melanogaster tyramine receptor cascade to plant essential oils

This paper reports the role of the tyramine (TA) receptor cascade in the insecticidal activity of plant essential oils. A TA receptor cDNA encoding a putative seven transmembrane domain G-protein coupled receptor was amplified from Drosophila melanogaster head cDNA phage library. The encoded protein contains 601 amino acids and has a sequence similar to other biogenic amine receptors. This protein was expressed in Drosophila S2 cells for radioligand binding studies with the ligand 3H-TA. Competitive binding studies comparing biogenic amines that could potentially function as endogenous ligands have demonstrated that this receptor had the highest affinity for TA (Ki=1.27 microM) followed by DL-octopamine, dopamine, serotonin and histamine. TA decreased the forskolin-increased cAMP levels (IC50=5.802 microM) and increased [Ca2+]i through the receptor expressed in S2 cells. The toxicity rank order of the tested plant essential oils against wild type D. melanogaster fly demonstrated a pattern similar to their effect on receptor binding activity and changes in cAMP level and [Ca2+]i. The toxicity of two of these chemicals was eliminated when tested against the TA receptor mutant (TyrRneo30) Drosophila strain. Therefore, the data indicates a correlation between cellular changes and insecticidal activity of tested plant essential oils, and suggests that the toxicity of at least two of these chemicals is mediated through the TA receptor.     

Locomotor activity is regulated by D2-like receptors in Drosophila: an anatomic and functional analysis

In mammals, dopamine 2-like receptors are expressed in distinct pathways within the central nervous system, as well as in peripheral tissues. Selected neuronal D2-like receptors play a critical role in modulating locomotor activity and, as such, represent an important therapeutic target (e.g. in Parkinson's disease). Previous studies have established that proteins required for dopamine (DA) neurotransmission are highly conserved between mammals and the fruit fly Drosophila melanogaster. These include a fly dopamine 2-like receptor (DD2R; Hearn et al. PNAS 2002 99(22):14554) that has structural and pharmacologic similarity to the human D2-like (D2R). In the current study, we define the spatial expression pattern of DD2R, and functionally characterize flies with reduced DD2 receptor levels. We show that DD2R is expressed in the larval and adult nervous systems, in cell groups that include the Ap-let cohort of peptidergic neurons, as well as in peripheral tissues including the gut and Malpighian tubules. To examine DD2R function in vivo, we generated RNA-interference (RNAi) flies with reduced DD2R expression. Behavioral analysis revealed that these flies show significantly decreased locomotor activity, similar to the phenotype observed in mammals with reduced D2R expression. The fly RNAi phenotype can be rescued by administration of the DD2R synthetic agonist bromocriptine, indicating specificity for the RNAi effect. These results suggest Drosophila as a useful system for future studies aimed at identifying modifiers of dopaminergic signaling/locomotor function.     

2,8-Disubstituted adenosine derivatives as partial agonists for the adenosine A2A receptor

Novel 2,8-disubstituted adenosine derivatives were synthesized in good overall yields starting from 2-iodoadenosine. Binding affinities were determined for rat adenosine A(1) and A(2A) receptors and human A(3) receptors. Some compounds displayed good adenosine A(2A) receptor affinities, with most of the 2-(1-hexynyl)- and 2-[(E)-1-hexenyl]-substituted derivatives having K(i) values in the nanomolar range. Although the introduction of an 8-alkylamino substituents decreased the affinity for the adenosine A(2A) receptor somewhat, the selectivity for this receptor compared to A(3) was improved significantly. The 8-methylamino (12) and 8-propylamino (14) derivatives of 2-(1-hexynyl)adenosine (3), showed reasonable A(2A) receptor affinities with K(i) values of 115 and 82nM, respectively, and were 49- and 26-fold selective for the adenosine A(2A) receptor compared to the A(3) receptor. The compounds were also evaluated for their ability to stimulate the cAMP production in CHO cells expressing the human adenosine A(2A) receptor. 2-(1-Hexynyl)adenosine (3) and 2-[(E)-1-hexenyl]adenosine (4) both showed submaximal levels of produced cAMP, compared to the reference full agonist CGS 21680, and thus behaved as partial agonists. Most 8-alkylamino-substituted derivatives of 3, displayed similar cAMP production as 3, and behaved as partial agonists as well. Introduction of alkylamino groups at the 8-position of 4, showed a slight reduction of the efficacy compared to 4, and these compounds were partial agonists also.     

VPAC(1) receptors have different agonist efficacy profiles on membrane and intact cells

The vasoactive intestinal peptide receptor VPAC(1) is preferentially coupled to G(alpha s) protein but also increases [Ca(2+)](i) through interaction with G(alpha i)/G(alpha q) protein. We evaluated a panel of full, partial and null agonists for their capability to stimulate adenylate cyclase activity in both intact cells and membrane and [Ca(2+)](i) in intact cells transfected with the reporter gene aequorin. In intact cells, the agonists efficacy for cAMP and calcium increase were well, but not linearly correlated: VPAC(1) receptors activated G(alpha s) protein more efficiently but with the same pharmacological profile as the other G proteins. In contrast, there was a difference between cAMP increase in intact and broken cell membranes: EC(50) values were generally lower in intact cells whereas the efficacy was higher. There was, however, no correlation between the shift in the EC(50) value and the intrinsic activity. Of interest, the (4-28) fragment, a reported antagonist on cell membrane, was a full agonist in intact cells. We concluded that the active states of the VPAC(1) receptor resulting from the coupling to different effector are undistinguishable by the VIP analogs tested but that receptor properties are different when evaluated in intact cells or cell membranes.     

Modulation of adenosine receptor affinity and intrinsic efficacy in adenine nucleosides substituted at the 2-position

We studied the structural determinants of binding affinity and efficacy of adenosine receptor (AR) agonists. Substituents at the 2-position of adenosine were combined with N(6)-substitutions known to enhance human A(3)AR affinity. Selectivity of binding of the analogues and their functional effects on cAMP production were studied using recombinant human A(1), A(2A), A(2B), and A(3)ARs. Mainly sterically small substituents at the 2-position modulated both the affinity and intrinsic efficacy at all subtypes. The 2-cyano group decreased hA(3)AR affinity and efficacy in the cases of N(6)-(3-iodobenzyl) and N(6)-(trans-2-phenyl-1-cyclopropyl), for which a full A(3)AR agonist was converted into a selective antagonist; the 2-cyano-N(6)-methyl analogue was a full A(3)AR agonist. The combination of N(6)-benzyl and various 2-substitutions (chloro, trifluoromethyl, and cyano) resulted in reduced efficacy at the A(1)AR. The environment surrounding the 2-position within the putative A(3)AR binding site was explored using rhodopsin-based homology modeling and ligand docking.     

A sleep-promoting role for the Drosophila serotonin receptor 1A

BACKGROUND: Although sleep is an important process essential for life, its regulation is poorly understood. The recently developed Drosophila model for sleep provides a powerful system to genetically and pharmacologically identify molecules that regulate sleep. Serotonin is an important neurotransmitter known to affect many behaviors, but its role in sleep remains controversial. RESULTS: We generated or obtained flies with genetically altered expression of each of three Drosophila serotonin receptor subtypes (d5-HT1A, d5-HT1B, and d5-HT2) and assayed them for baseline sleep phenotypes. The data indicated a sleep-regulating role for the d5-HT1A receptor. d5-HT1A mutant flies had short and fragmented sleep, which was rescued by expressing the receptor in adult mushroom bodies, a structure associated with learning and memory in Drosophila. Neither the d5-HT2 receptor nor the d5-HT1B receptor, which was previously implicated in circadian regulation, had any effect on baseline sleep, indicating that serotonin affects sleep and circadian rhythms through distinct receptors. Elevating serotonin levels, either pharmacologically or genetically, enhanced sleep in wild-type flies. In addition, serotonin promoted sleep in some short-sleep mutants, suggesting that it can compensate for some sleep deficits. CONCLUSIONS: These data show that serotonin promotes baseline sleep in Drosophila. They also link the regulation of sleep behavior by serotonin to a specific receptor in a distinct region of the fly brain.     

Inhibition of human airway smooth muscle cell proliferation by beta 2-adrenergic receptors and cAMP is PKA independent: evidence for EPAC involvement

Mechanisms by which beta-adrenergic receptor (beta AR) agonists inhibit proliferation of human airway smooth muscle (HASM) cells were investigated because of their potential relevance to smooth muscle hyperplasia in asthma. We hypothesized that beta AR agonists would inhibit mitogenesis in HASM cells via the beta 2AR, an increase in cAMP, and PKA activation. HASM cells were treated for 24 h with various agents and then analyzed for [3H]thymidine incorporation as a measure of cell proliferation. EGF stimulated proliferation by approximately 10-fold. The nonselective beta AR agonist isoproterenol and the beta 2AR-selective agonists albuterol and salmeterol inhibited EGF-stimulated proliferation by more than 50%, with half-maximal effects at 4.8 nM, 110 nM, and 6.7 nM, respectively. A beta 2AR-selective antagonist inhibited the isoproterenol effect with 100-fold greater potency than a beta 1AR-selective antagonist, confirming beta 2AR involvement in the inhibition of proliferation. The cAMP-elevating agents PGE2 and forskolin decreased EGF-induced proliferation, suggesting cAMP as the mediator. beta 2AR agonists and forskolin also inhibited proliferation stimulated by lysophosphatidic acid (LPA) as well as the synergistic proliferation stimulated by LPA+EGF. Importantly, PKA-selective cAMP analogs did not inhibit proliferation at concentrations that maximally activated PKA (10-100 microM), whereas a cAMP analog selective for the exchange protein directly activated by cAMP (EPAC), 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, maximally inhibited proliferation at a concentration that did not activate PKA (10 microM). These data show that beta 2AR agonists and other cAMP-elevating agents decrease proliferation in HASM cells via a PKA-independent mechanism, and they provide pharmacological evidence for involvement of EPAC or an EPAC-like cAMP effector protein instead.     

Endogenous Gs-coupled receptors in smooth muscle exhibit differential susceptibility to GRK2/3-mediated desensitization

Although G protein-coupled receptor (GPCR) kinases (GRKs) have been shown to mediate desensitization of numerous GPCRs in studies using cellular expression systems, their function under physiological conditions is less well understood. In the current study, we employed various strategies to assess the effect of inhibiting endogenous GRK2/3 on signaling and function of endogenously expressed G s-coupled receptors in human airway smooth muscle (ASM) cells. GRK2/3 inhibition by expression of a Gbetagamma sequestrant, a GRK2/3 dominant-negative mutant, or siRNA-mediated knockdown increased intracellular cAMP accumulation mediated via beta-agonist stimulation of the beta-2-adrenergic receptor (beta 2AR). Conversely, neither 5'-( N-ethylcarboxamido)-adenosine (NECA; activating the A2b adenosine receptor) nor prostaglandin E2 (PGE 2; activating EP2 or EP4 receptors)-stimulated cAMP was significantly increased by GRK2/3 inhibition. Selective knockdown using siRNA suggested the majority of PGE 2-stimulated cAMP in ASM was mediated by the EP2 receptor. Although a minor role for EP3 receptors in influencing PGE 2-mediated cAMP was determined, the GRK2/3-resistant nature of EP2 receptor signaling in ASM was confirmed using the EP2-selective agonist butaprost. Somewhat surprisingly, GRK2/3 inhibition did not augment the inhibitory effect of the beta-agonist on mitogen-stimulated increases in ASM growth. These findings demonstrate that with respect to G s-coupled receptors in ASM, GRK2/3 selectively attenuates beta 2AR signaling, yet relief of GRK2/3-dependent beta 2AR desensitization does not influence at least one important physiological function of the receptor.