Activation of endogenous GABAA channels on airway smooth muscle potentiates isoproterenol-mediated relaxation

Reactive airway disease predisposes patients to episodes of acute smooth muscle mediated bronchoconstriction. We have for the first time recently demonstrated the expression and function of endogenous ionotropic GABA(A) channels on airway smooth muscle cells. We questioned whether endogenous GABA(A) channels on airway smooth muscle could augment beta-agonist-mediated relaxation. Guinea pig tracheal rings or human bronchial airway smooth muscles were equilibrated in organ baths with continuous digital tension recordings. After pretreatment with or without the selective GABA(A) antagonist gabazine (100 muM), airway muscle was contracted with acetylcholine or beta-ala neurokinin A, followed by relaxation induced by cumulatively increasing concentrations of isoproterenol (1 nM to 1 muM) in the absence or presence of the selective GABA(A) agonist muscimol (10-100 muM). In separate experiments, guinea pig tracheal rings were pretreated with the large conductance K(Ca) channel blocker iberiotoxin (100 nM) after an EC(50) contraction with acetylcholine but before cumulatively increasing concentrations of isoproterenol (1 nM to 1 uM) in the absence or presence of muscimol (100 uM). GABA(A) activation potentiated the relaxant effects of isoproterenol after an acetylcholine or tachykinin-induced contraction in guinea pig tracheal rings or an acetylcholine-induced contraction in human endobronchial smooth muscle. This muscimol-induced potentiation of relaxation was abolished by gabazine pretreatment but persisted after blockade of the maxi K(Ca) channel. Selective activation of endogenous GABA(A) receptors significantly augments beta-agonist-mediated relaxation of guinea pig and human airway smooth muscle, which may have important therapeutic implications for patients in severe bronchospasm.     

The dopamine D1 receptor is expressed and facilitates relaxation in airway smooth muscle

Background

Dopamine signaling is mediated by G_s protein-coupled “D₁-like” receptors (D₁ and D₅) and G_i-coupled “D₂-like” receptors (D_2-4). In asthmatic patients, inhaled dopamine induces bronchodilation. Although the G_i-coupled dopamine D₂ receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle (ASM) cells, the G_s-coupled dopamine D₁-like receptor subtypes have never been identified on these cells. Activation of G_s-coupled receptors stimulates cyclic AMP (cAMP) production through the stimulation of adenylyl cyclase, which promotes ASM relaxation. We questioned whether the dopamine D₁-like receptor is expressed on ASM, and modulates its function through G_s-coupling.

Methods

The mRNA and protein expression of dopamine D₁-like receptor subtypes in both native human and guinea pig ASM tissue and cultured human ASM (HASM) cells was measured. To characterize the stimulation of cAMP through the dopamine D₁ receptor, HASM cells were treated with dopamine or the dopamine D₁-like receptor agonists ({"type":"entrez-nucleotide","attrs":{"text":"A68930","term_id":"4759850","term_text":"A68930"}}A68930 or {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393) before cAMP measurements. To evaluate whether the activation of dopamine D₁ receptor induces ASM relaxation, guinea pig tracheal rings suspended under isometric tension in organ baths were treated with cumulatively increasing concentrations of dopamine or {"type":"entrez-nucleotide","attrs":{"text":"A68930","term_id":"4759850","term_text":"A68930"}}A68930, following an acetylcholine-induced contraction with or without the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the large-conductance calcium-activated potassium (BK_Ca) channel blocker iberiotoxin, or the exchange proteins directly activated by cAMP (Epac) antagonist NSC45576.

Results

Messenger RNA encoding the dopamine D₁ and D₅ receptors were detected in native human ASM tissue and cultured HASM cells. Immunoblots confirmed the protein expression of the dopamine D₁ receptor in both native human and guinea pig ASM tissue and cultured HASM cells. The dopamine D₁ receptor was also immunohistochemically localized to both human and guinea pig ASM. The dopamine D₁-like receptor agonists stimulated cAMP production in HASM cells, which was reversed by the selective dopamine D₁-like receptor antagonists {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 or {"type":"entrez-protein","attrs":{"text":"SCH39166","term_id":"1052842517","term_text":"SCH39166"}}SCH39166. {"type":"entrez-nucleotide","attrs":{"text":"A68930","term_id":"4759850","term_text":"A68930"}}A68930 relaxed acetylcholine-contracted guinea pig tracheal rings, which was attenuated by Rp-cAMPS but not by iberiotoxin or NSC45576.

Conclusions

These results demonstrate that the dopamine D₁ receptors are expressed on ASM and regulate smooth muscle force via cAMP activation of PKA, and offer a novel target for therapeutic relaxation of ASM.     

Functional TRPV4 channels are expressed in human airway smooth muscle cells

Hypotonic stimulation induces airway constriction in normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca(2+) influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4alpha-phorbol 12,13-didecanoate (4-alphaPDD), a TRPV4 ligand, increased intracellular Ca(2+) level only when Ca(2+) was present in the extracellular solution. The 4-alphaPDD-induced Ca(2+) response was inhibited by ruthenium red (1 microM), a known TRPV4 inhibitor, but not by capsazepine (1 microM), a TRPV1 antagonist, indicating that 4-alphaPDD-induced Ca(2+) response is mediated by TRPV4. Verapamil (10 microM), an L-type voltage-gated Ca(2+) channel inhibitor, had no effect on the 4-alphaPDD-induced Ca(2+) response, excluding the involvement of L-type Ca(2+) channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca(2+) channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca(2+) channel inhibitor nifedipine (1 microM) or by the TRPV1 inhibitor capsazepine (1 microM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.     

Mechanisms of airway smooth muscle relaxation during maturation

Greater airway responsiveness in healthy juveniles is considered a factor in the higher asthma prevalence at a young age compared with adults. We have developed a guinea pig maturational model that utilizes tracheal strips from 1-week-, 3-week-, and 3-month-old guinea pigs to study the role of airway smooth muscle (ASM) in juvenile airway hyperresponsiveness. Because a reduced ability of ASM to spontaneously relax may contribute to airway hyperresponsiveness by maintaining bronchospasm and thus high airway resistance, we have employed this model to study ASM spontaneous relaxation during electrical field stimulation (EFS). Since relaxation during EFS had been neither described nor quantified during maturation, we developed new indices that allowed an appropriate comparison of the relaxing response from strips of different age animals. Using these indices we found that, whereas strips from adult animals relax to a level of tension similar to that found in the absence of stimulation, this ability to spontaneously relax is essentially absent in trachealis from infant animals. These results confirmed that maturation of ASM relaxation may play a role in juvenile airway hyperresponsiveness and that our maturational model is suitable to study the mechanisms regulating spontaneous relaxation in physiological conditions. We investigated the role of prostanoids in ASM relaxation and showed that cyclooxygenase inhibition increases relaxation in infant ASM to levels similar to adults. These results suggest that prostanoids regulate the ability of ASM to spontaneously relax, i.e., they reduce relaxation. We have produced preliminary data suggesting a maturational change in the level of prostanoids. Moreover, the possible action of acetylcholinesterase on maturation of ASM relaxation is discussed here on the basis of a preliminary study. We suggest that impairment of ASM relaxation likely contributes to increased airway responsiveness.     

Isotonic relaxation of control and sensitized airway smooth muscle

Smooth muscle relaxation has most often been studied in isometric mode. However, this only tells us about the stiffness properties of the bronchial wall and thus only about wall capacitative properties. It tells us little about airflow. To study the latter, which of course is the meaningful parameter in regulation of ventilation and in asthma, we studied isotonic shortening of bronchial smooth muscle (BSM) strips. Failure of BSM to relax could be another important factor in maintaining high airway resistance. To analyze relaxation curves, we developed an index of isotonic relaxation, t1/2(P, lCE), which is the half-time for relaxation that is independent of muscle load (P) and of initial contractile element length (lCE). This index was measured in curves of relaxation initiated at 2 s (normally cycling crossbridges) and at 10 s (latch-bridges). At 10 s no difference was seen for adjusted t1/2(P, lCE) between curves obtained from control and sensitized BSM, (8.38 +/- 0.92 s vs. 7.78 +/- 0.93 s, respectively). At 2 s the half-time was almost doubled in the sensitized BSM (6.98 +/- 0.01 s (control) vs. 12.74 +/- 2.5 s (sensitized)). Thus, changes in isotonic relaxation are only seen during early contraction. Using zero load clamps, we monitored the time course of velocity during relaxation and noted that it varied according to 3 phases. The first phase (phase i) immediately followed cessation of electrical field stimulation (EFS) at 10 s and showed almost the same velocity as during the latter 1/3 of shortening; the second phase (phase ii) was linear in shape and is associated with zero load velocity, we speculate it could stem from elastic recoil of the cells' internal resistor; and the third phase (phase iii) was convex downwards. The zero load velocities in phase iii showed a surprising spontaneous increase suggesting reactivation of the muscle. Measurements of intracellular calcium (Fura-2 study) and of phosphorylation of the 20 kDa myosin light chain showed simultaneous increments, indicating phase iii represented an active process. Studies are under way to determine what changes occur in these 3 phases in a sensitized muscle. And of course, in the context of this conference, just what role the plastic properties of the muscle play in relaxation requires serious consideration.     

S-nitrosoglutathione breakdown prevents airway smooth muscle relaxation in the guinea pig

Airway levels of the endogenous bronchodilator S-nitrosoglutathione (GSNO) are low in children with near-fatal asthma. We hypothesized that GSNO could be broken down in the lung and that this catabolism could inhibit airway smooth muscle relaxation. In our experiments, GSNO was broken down by guinea pig lung homogenates, particularly after ovalbumin sensitization (OS). Two lung protein fractions had catabolic activity. One was NADPH dependent and was more active after OS. The other was NADPH independent and was partially inhibited by aurothioglucose. Guinea pig lung tissue protein fractions with GSNO catabolic activity inhibited GSNO-mediated guinea pig tracheal ring relaxation. The relaxant effect of GSNO was partially restored by aurothioglucose. These observations suggest that catabolism of GSNO in the guinea pig 1) is mediated by lung proteins, 2) is partially upregulated after OS, and 3) may contribute to increased airway smooth muscle tone. We speculate that enzymatic breakdown of GSNO in the lung could contribute to asthma pathophysiology by inhibiting the beneficial effects of GSNO, including its effect on airway smooth muscle tone.     

cGMP-independent mechanism of airway smooth muscle relaxation induced by S-nitrosoglutathione

This study tested the hypothesis that the NO donorS-nitrosoglutathione (GSNO) relaxescanine tracheal smooth muscle (CTSM) in part by a cGMP-independentprocess that involves reversible oxidation of intracellular thiols.GSNO caused a concentration-dependent relaxation in ACh-contractedstrips (EC₅₀ ~1.2 µM)accompanied by a concentration-dependent increase in cytosolic cGMPconcentration ([cGMP]_i). Thesoluble guanylate cyclase inhibitor methylene blue prevented theincrease in [cGMP]_iinduced by 1 and 10 µM GSNO, but isometric force decreased by 10 ± 4 and 55 ± 3%, respectively. After recovery of[cGMP]_i to baseline,GSNO-induced relaxation persisted during continuous ACh stimulation.Dithiothreitol caused a rapid recovery of isometric force to valuessimilar to those obtained with ACh alone in these strips. We concludethat GSNO relaxes CTSM contracted by ACh in part by oxidation ofintracellular protein thiols.

     

Facilitation of isoproterenol induced airway smooth muscle relaxation by nifedipine

The facilitating effect of nifedipine on isoproterenol induced airway smooth muscle relaxation was studied in guinea pig tracheas. For isometric force measurement, 4 mm tracheal cylinders were suspended in incubation chambers in oxygenated physiologic medium. After 90 minutes of equilibration under 2 grams resting tension, at a temperature of 37 degrees C and pH of 7.4, concentration response curves for isoproterenol were performed with and without the addition of a 1 X 10(-5)M nifedipine dose. The experiments were then repeated using tissues precontracted with histamine. Our data show that in the nifedipine pretreated tissues, the EC50 of isoproterenol is shifted to the left (p less than 0.05) probably due to further reduction in cytosolic calcium by nifedipine. Our findings suggest that nifedipine might have a role in the treatment of asthma and obstructive airway disease.     

Epac as a novel effector of airway smooth muscle relaxation

Dysfunctional regulation of airway smooth muscle tone is a feature of obstructive airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle contraction is directly associated with changes in the phosphorylation of myosin light chain (MLC), which is increased by Rho and decreased by Rac. Although cyclic adenosine monophosphate (cAMP)‐elevating agents are believed to relieve bronchoconstriction mainly via activation of protein kinase A (PKA), here we addressed the role of the novel cAMP‐mediated exchange protein Epac in the regulation of airway smooth muscle tone. Isometric tension measurements showed that specific activation of Epac led to relaxation of guinea pig tracheal preparations pre‐contracted with methacholine, independently of PKA. In airway smooth muscle cells, Epac activation reduced methacholine‐induced MLC phosphorylation. Moreover, when Epac was stimulated, we observed a decreased methacholine‐induced RhoA activation, measured by both stress fibre formation and pull‐down assay whereas the same Epac activation prevented methacholine‐induced Rac1 inhibition measured by pull‐down assay. Epac‐driven inhibition of both methacholine‐induced muscle contraction by Toxin B‐1470, and MLC phosphorylation by the Rac1‐inhibitor NSC23766, were significantly attenuated, confirming the importance of Rac1 in Epac‐mediated relaxation. Importantly, human airway smooth muscle tissue also expresses Epac, and Epac activation both relaxed pre‐contracted human tracheal preparations and decreased MLC phosphorylation. Collectively, we show that activation of Epac relaxes airway smooth muscle by decreasing MLC phosphorylation by skewing the balance of RhoA/Rac1 activation towards Rac1. Therefore, activation of Epac may have therapeutical potential in the treatment of obstructive airway diseases.     

Signaling and regulation of G protein-coupled receptors in airway smooth muscle

Signaling through G protein-coupled receptors (GPCRs) mediates numerous airway smooth muscle (ASM) functions including contraction, growth, and "synthetic" functions that orchestrate airway inflammation and promote remodeling of airway architecture. In this review we provide a comprehensive overview of the GPCRs that have been identified in ASM cells, and discuss the extent to which signaling via these GPCRs has been characterized and linked to distinct ASM functions. In addition, we examine the role of GPCR signaling and its regulation in asthma and asthma treatment, and suggest an integrative model whereby an imbalance of GPCR-derived signals in ASM cells contributes to the asthmatic state.     

'Proliferative' and 'synthetic' airway smooth muscle cells are overlapping populations

The extension of airway smooth muscle cell (ASMC) functions, from just contractile, to synthetic and/or proliferative states, is an important component of airway remodelling and inflammation in asthma. Whereas all these functions have been demonstrated in ASM, currently, it is not known whether ASMC can be differentiated on the basis of their proliferative and synthetic functions. We used flow-cytometric techniques to determine, first, whether human ASMC are phenotypically heterogenous with regard to their secretory function, and second, the proliferative status of secretory cells. ASMC were induced to synthesize GM-CSF by stimulation with IL-1beta and TNF-alpha followed by 10% human serum. Flow-cytometric detection of intracellular GM-CSF revealed that only a proportion of cells in culture (approximately 20-60%) synthesize GM-CSF. To determine the proliferative status of GM-CSF producing cells, ASMC were pretreated with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE), a fluorescein based dye used to track cell division, prior to cytokine/serum stimulation. Simultaneous analysis of intracellular GM-CSF and CFSE revealed that GM-CSF producing cells were present in both the divided and undivided ASMC populations. Thus, cytokine production and proliferation occurred in overlapping ASMC populations and prior progression through the cell cycle was not essential for ASMC cytokine production.     

cAMP, myosin dephosphorylation, and isometric relaxation of airway smooth muscle

The temporal relationships among increases in adenosine 3',5'-cyclic monophosphate (cAMP) levels, myosin dephosphorylation, and relaxation were investigated to clarify the mechanisms of airway muscle relaxation. Canine tracheal muscles isometrically contracted (82% of maximum force) with 10(-6) M methacholine were relaxed by adding either 4 x 10(-7) M atropine or 4 x 10(-5) M forskolin. Atropine had no effect on cAMP levels; myosin phosphorylation and force, however, decayed at the same rates and these two parameters returned to their basal pre-methacholine levels within 5 min. Forskolin treatment results in about a 10-fold increase in cAMP levels; myosin phosphorylation and force decayed simultaneously to their respective steady-state levels by 10 min but neither parameter returned to its pre-methacholine level. The addition of forskolin to muscles maximally contracted with 10(-4) M methacholine leads to about a 30-fold increase in cAMP levels. However, there are minimal decreases in myosin phosphorylation and force in these muscles. Thus myosin dephosphorylation appears to be essential for airway muscle relaxation, whereas an increase in cAMP in the absence of myosin dephosphorylation is insufficient to cause relaxation. Moreover, myosin dephosphorylation appears to be a common step in the cAMP-independent and cAMP-dependent mechanisms for airway muscle relaxation.     

Caveolae facilitate muscarinic receptor-mediated intracellular Ca2+ mobilization and contraction in airway smooth muscle

Contractile responses of airway smooth muscle (ASM) determine airway resistance in health and disease. Caveolae microdomains in the plasma membrane are marked by caveolin proteins and are abundant in contractile smooth muscle in association with nanospaces involved in Ca(2+) homeostasis. Caveolin-1 can modulate localization and activity of signaling proteins, including trimeric G proteins, via a scaffolding domain. We investigated the role of caveolae in contraction and intracellular Ca(2+) ([Ca(2+)](i)) mobilization of ASM induced by the physiological muscarinic receptor agonist, acetylcholine (ACh). Human and canine ASM tissues and cells predominantly express caveolin-1. Muscarinic M(3) receptors (M(3)R) and Galpha(q/11) cofractionate with caveolin-1-rich membranes of ASM tissue. Caveolae disruption with beta-cyclodextrin in canine tracheal strips reduced sensitivity but not maximum isometric force induced by ACh. In fura-2-loaded canine and human ASM cells, exposure to methyl-beta-cyclodextrin (mbetaCD) reduced sensitivity but not maximum [Ca(2+)](i) induced by ACh. In contrast, both parameters were reduced for the partial muscarinic agonist, pilocarpine. Fluorescence microscopy revealed that mbetaCD disrupted the colocalization of caveolae-1 and M(3)R, but [N-methyl-(3)H]scopolamine receptor-binding assay revealed no effect on muscarinic receptor availability or affinity. To dissect the role of caveolin-1 in ACh-induced [Ca(2+)](i) flux, we disrupted its binding to signaling proteins using either a cell-permeable caveolin-1 scaffolding domain peptide mimetic or by small interfering RNA knockdown. Similar to the effects of mbetaCD, direct targeting of caveolin-1 reduced sensitivity to ACh, but maximum [Ca(2+)](i) mobilization was unaffected. These results indicate caveolae and caveolin-1 facilitate [Ca(2+)](i) mobilization leading to ASM contraction induced by submaximal concentrations of ACh.     

Role of M2 muscarinic receptors in airway smooth muscle contraction

Airway smooth muscle expresses both M2 and M3 muscarinic receptors with the majority of the receptors of the M2 subtype. Activation of M3 receptors, which couple to Gq, initiates contraction of airway smooth muscle while activation of M2 receptors, which couple to Gi, inhibits beta-adrenergic mediated relaxation. Increased sensitivity to intracellular Ca2+ is an important mechanism for agonist-induced contraction of airway smooth muscle but the signal transduction pathways involved are uncertain. We studied Ca2+ sensitization by acetylcholine (ACh) and endothelin-1 (ET-1) in porcine tracheal smooth muscle by measuring contractions at constant [Ca2+] in strips permeabilized with Staphylococcal alpha-toxin. Both ACh and ET-1 contracted airway smooth muscle at constant [Ca2+]. Pretreatment with pertussis toxin for 18-20 hours reduced ACh contractions, but had no effect on those of ET-1 or GTPgammaS. We conclude that the M2 muscarinic receptor contributes to airway smooth muscle contraction at constant [Ca2+] via the heterotrimeric G-protein Gi.     

Activation of the midbrain periaqueductal gray induces airway smooth muscle relaxation.

In this study, we examined effects of chemical stimulation of the ventrolateral region of the midbrain periaqueductal gray (vl PAG) on airway smooth muscle tone. We observed that in anesthetized, paralyzed, and artificially ventilated ferrets, vl PAG stimulation elicited airway smooth muscle relaxation. To clarify the mechanisms underlying this observation, we examined the GABA-GABA(A) receptor signaling pathway by 1) examining the expression of GABA(A) receptors on airway-related vagal preganglionic neurons (AVPNs) located in the rostral nucleus ambiguus region (rNA), by use of receptor immunochemistry and confocal microscopy; 2) measuring GABA release within the rNA by using microdialysis; and 3) performing physiological experiments to determine the effects of selective blockade of GABA(A) receptors expressed by AVPNs in the rNA region on vl PAG-induced airway relaxation, thereby defining the role of the GABA(A) receptor subtype in this process. We observed that AVPNs located in the rNA region do express the GABA(A) receptor beta-subtype. In addition, we demonstrated that activation of vl PAG induced GABA release within the rNA region, and this release was associated with airway smooth muscle relaxation. Blockade of the GABA(A) receptor subtype expressed by AVPNs in the rNA by bicuculline diminished the inhibitory effects of vl PAG stimulation on airway smooth muscle tone. These data indicate, for the first time, that activation of vl PAG dilates the airways by a release of GABA and activation of GABA(A) receptors expressed by AVPNs.     

Arrestin specificity for G protein-coupled receptors in human airway smooth muscle.

Despite a widely accepted role of arrestins as "uncouplers" of G protein-coupled receptor (GPCR) signaling, few studies have demonstrated the ability of arrestins to affect second messenger generation by endogenously expressed receptors in intact cells. In this study we demonstrate arrestin specificity for endogenous GPCRs in primary cultures of human airway smooth muscle (HASM). Expression of arrestin-green fluorescent protein (ARR2-GFP or ARR3-GFP) chimeras in HASM significantly attenuated isoproterenol (beta(2)-adrenergic receptor (beta(2)AR)-mediated)- and 5'-(N-ethylcarboxamido)adenosine (A2b adenosine receptor-mediated)-stimulated cAMP production, with fluorescent microscopy demonstrating agonist-promoted redistribution of cellular ARR2-GFP into a punctate formation. Conversely, prostaglandin E(2) (PGE(2))-mediated cAMP production was unaffected by arrestin-GFP, and PGE(2) had little effect on arrestin-GFP distribution. The pharmacological profile of various selective EP receptor ligands suggested a predominantly EP2 receptor population in HASM. Further analysis in COS-1 cells revealed that ARR2-GFP expression increased agonist-promoted internalization of wild type beta(2)AR and EP4 receptors, whereas EP2 receptors remained resistant to internalization. However, expression of an arrestin whose binding to GPCRs is largely independent of receptor phosphorylation (ARR2(R169E)-GFP) enabled substantial agonist-promoted EP2 receptor internalization, increased beta(2)AR internalization to a greater extent than did ARR2-GFP, yet promoted EP4 receptor internalization to the same degree as did ARR2-GFP. Signaling via endogenous EP4 receptors in CHO-K1 cells was attenuated by ARR2-GFP expression, whereas ARR2(R169E)-GFP expression in HASM inhibited EP2 receptor-mediated cAMP production. These findings demonstrate differential effects of arrestins in altering endogenous GPCR signaling in a physiologically relevant cell type and reveal a variable dependence on receptor phosphorylation in dictating arrestin-receptor interaction.