Unraveling allostery within the angiotensin II type 1 receptor for Gαq and ß-arrestin coupling.

G protein-coupled receptors engage both G proteins and ß-arrestins, and their coupling can be biased by ligands and mutations. Here, to resolve structural elements and mechanisms underlying effector coupling to the angiotensin II (AngII) type 1 receptor (AT1R), we combined alanine scanning mutagenesis of the entire sequence of the receptor with pharmacological profiling of Gαq and ß-arrestin engagement to mutant receptors and molecular dynamics simulations. We showed that Gαq coupling to AT1R involved a large number of residues spread across the receptor, whereas fewer structural regions of the receptor contributed to ß-arrestin coupling regulation. Residue stretches in transmembrane domain 4 conferred ß-arrestin bias and represented an important structural element in AT1R for functional selectivity. Furthermore, we identified allosteric small-molecule binding sites that were enclosed by communities of residues that produced biased signaling when mutated. Last, we showed that allosteric communication within AT1R emanating from the Gαq coupling site spread beyond the orthosteric AngII-binding site and across different regions of the receptor, including currently unresolved structural regions. Our findings reveal structural elements and mechanisms within AT1R that bias Gαq and ß-arrestin coupling and that could be harnessed to design biased receptors for research purposes and to develop allosteric modulators.

Dynamic spatiotemporal determinants modulate GPCR:G protein coupling selectivity and promiscuity.

Recent studies have shown that G protein coupled receptors (GPCRs) show selective and promiscuous coupling to different Gα protein subfamilies and yet the mechanisms of the range of coupling preferences remain unclear. Here, we use Molecular Dynamics (MD) simulations on ten GPCR:G protein complexes and show that the location (spatial) and duration (temporal) of intermolecular contacts at the GPCR:Gα protein interface play a critical role in how GPCRs selectively interact with G proteins. We identify that some GPCR:G protein interface contacts are common across Gα subfamilies and others specific to Gα subfamilies. Using large scale data analysis techniques on the MD simulation snapshots we derive a spatio-temporal code for contacts that confer G protein selective coupling and validated these contacts using G protein activation BRET assays. Our results demonstrate that promiscuous GPCRs show persistent sampling of the common contacts more than G protein specific contacts. These findings suggest that GPCRs maintain contact with G proteins through a common central interface, while the selectivity comes from G protein specific contacts at the periphery of the interface.

Allosteric modulation of GPCR-induced ß-arrestin trafficking and signaling by a synthetic intrabody.

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of ß-arrestin (ßarr) recruitment and trafficking. For several GPCRs such as the vasopressin receptor subtype 2 (V2R), agonist-stimulation first drives the translocation of ßarrs to the plasma membrane, followed by endosomal trafficking, which is generally considered to be orchestrated by multiple phosphorylation sites. We have previously shown that mutation of a single phosphorylation site in the V2R (i.e., V2RT360A) results in near-complete loss of ßarr translocation to endosomes despite robust recruitment to the plasma membrane, and compromised ERK1/2 activation. Here, we discover that a synthetic intrabody (Ib30), which selectively recognizes activated ßarr1, efficiently rescues the endosomal trafficking of ßarr1 and ERK1/2 activation for V2RT360A. Molecular dynamics simulations reveal that Ib30 enriches active-like ßarr1 conformation with respect to the inter-domain rotation, and cellular assays demonstrate that it also enhances ßarr1-ß2-adaptin interaction. Our data provide an experimental framework to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can be potentially integrated in the paradigm of GPCR-targeted drug discovery.

Standardized Cannabis Smoke Extract Induces Inflammation in Human Lung Fibroblasts.

Cannabis (marijuana) is the most commonly used illicit product in the world and is the second most smoked plant after tobacco. There has been a rapid increase in the number of countries legalizing cannabis for both recreational and medicinal purposes. Smoking cannabis in the form of a joint is the most common mode of cannabis consumption. Combustion of cannabis smoke generates many of the same chemicals as tobacco smoke. Although the impact of tobacco smoke on respiratory health is well-known, the consequence of cannabis smoke on the respiratory system and, in particular, the inflammatory response is unclear. Besides the combustion products present in cannabis smoke, cannabis also contains cannabinoids including Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). These compounds are hydrophobic and not present in aqueous solutions. In order to understand the impact of cannabis smoke on pathological mechanisms associated with adverse respiratory outcomes, the development of in vitro surrogates of cannabis smoke exposure is needed. Therefore, we developed a standardized protocol for the generation of cannabis smoke extract (CaSE) to investigate its effect on cellular mechanisms in vitro. First, we determined the concentration of Δ9-THC, one of the major cannabinoids, by ELISA and found that addition of methanol to the cell culture media during generation of the aqueous smoke extract significantly increased the amount of Δ9-THC. We also observed by LC-MS/MS that CaSE preparation with methanol contains CBD. Using a functional assay in cells for CB1 receptors, the major target of cannabinoids, we found that this CaSE contains Δ9-THC which activates CB1 receptors. Finally, this standardized preparation of CaSE induces an inflammatory response in human lung fibroblasts. This study provides an optimized protocol for aqueous CaSE preparation containing biologically active cannabinoids that can be used for in vitro experimentation of cannabis smoke and its potential impact on various indices of pulmonary health.

Discovery of a dual Ras and ARF6 inhibitor from a GPCR endocytosis screen.

Internalization and intracellular trafficking of G protein-coupled receptors (GPCRs) play pivotal roles in cell responsiveness. Dysregulation in receptor trafficking can lead to aberrant signaling and cell behavior. Here, using an endosomal BRET-based assay in a high-throughput screen with the prototypical GPCR angiotensin II type 1 receptor (AT1R), we sought to identify receptor trafficking inhibitors from a library of ~115,000 small molecules. We identified a novel dual Ras and ARF6 inhibitor, which we named Rasarfin, that blocks agonist-mediated internalization of AT1R and other GPCRs. Rasarfin also potently inhibits agonist-induced ERK1/2 signaling by GPCRs, and MAPK and Akt signaling by EGFR, as well as prevents cancer cell proliferation. In silico modeling and in vitro studies reveal a unique binding modality of Rasarfin within the SOS-binding domain of Ras. Our findings unveil a class of dual small G protein inhibitors for receptor trafficking and signaling, useful for the inhibition of oncogenic cellular responses.

Pharmacological Characterization of the Imipridone Anticancer Drug ONC201 Reveals a Negative Allosteric Mechanism of Action at the D2 Dopamine Receptor.

ONC201 is a first-in-class imipridone compound that is in clinical trials for the treatment of high-grade gliomas and other advanced cancers. Recent studies identified that ONC201 antagonizes D2-like dopamine receptors at therapeutically relevant concentrations. In the current study, characterization of ONC201 using radioligand binding and multiple functional assays revealed that it was a full antagonist of the D2 and D3 receptors (D2R and D3R) with low micromolar potencies, similar to its potency for antiproliferative effects. Curve-shift experiments using D2R-mediated ß-arrestin recruitment and cAMP assays revealed that ONC201 exhibited a mixed form of antagonism. An operational model of allostery was used to analyze these data, which suggested that the predominant modulatory effect of ONC201 was on dopamine efficacy with little to no effect on dopamine affinity. To investigate how ONC201 binds to the D2R, we employed scanning mutagenesis coupled with a D2R-mediated calcium efflux assay. Eight residues were identified as being important for ONC201's functional antagonism of the D2R. Mutation of these residues followed by assessing ONC201 antagonism in multiple signaling assays highlighted specific residues involved in ONC201 binding. Together with computational modeling and simulation studies, our results suggest that ONC201 interacts with the D2R in a bitopic manner where the imipridone core of the molecule protrudes into the orthosteric binding site, but does not compete with dopamine, whereas a secondary phenyl ring engages an allosteric binding pocket that may be associated with negative modulation of receptor activity. SIGNIFICANCE STATEMENT: ONC201 is a novel antagonist of the D2 dopamine receptor with demonstrated efficacy in the treatment of various cancers, especially high-grade glioma. This study demonstrates that ONC201 antagonizes the D2 receptor with novel bitopic and negative allosteric mechanisms of action, which may explain its high selectivity and some of its clinical anticancer properties that are distinct from other D2 receptor antagonists widely used for the treatment of schizophrenia and other neuropsychiatric disorders.

Angiotensin II type 1 receptor variants alter endosomal receptor-ß-arrestin complex stability and MAPK activation.

The angiotensin II (AngII) type 1 receptor (AT1R), a member of the G protein-coupled receptor (GPCR) family, signals through G proteins and ß-arrestins, which act as adaptors to regulate AT1R internalization and mitogen-activated protein kinase (MAPK) ERK1/2 activation. ß-arrestin-dependent ERK1/2 regulation is the subject of important studies because its spatiotemporal control remains poorly understood for many GPCRs, including AT1R. To study the link between ß-arrestin-dependent trafficking and ERK1/2 signaling, we investigated three naturally occurring AT1R variants that show distinct receptor-ß-arrestin interactions: A163T, T282M, and C289W. Using bioluminescence resonance energy transfer (BRET)-based and conformational fluorescein arsenical hairpin-BRET sensors coupled with high-resolution fluorescence microscopy, we show that all AT1R variants form complexes with ß-arrestin2 at the plasma membrane and efficiently internalize into endosomes upon AngII stimulation. However, mutant receptors imposed distinct conformations in ß-arrestin2 and differentially impacted endosomal trafficking and MAPK signaling. Notably, T282M accumulated in endosomes, but its ability to form stable complexes following internalization was reduced, markedly impairing its ability to co-traffic with ß-arrestin2. We also found that despite ß-arrestin2 overexpression, T282M's and C289W's residency with ß-arrestin2 in endosomes was greatly reduced, leading to decreased ß-arrestin-dependent ERK1/2 activation, faster recycling of receptors to the plasma membrane, and impaired AngII-mediated proliferation. Our findings reveal that naturally occurring AT1R variants alter the patterns of receptor/ß-arrestin2 trafficking and suggest conformationally dependent ß-arrestin-mediated MAPK activation as well as endosomal receptor-ß-arrestin complex stabilization in the mitogenic response of AT1R.

Signal profiling of the ß1AR reveals coupling to novel signalling pathways and distinct phenotypic responses mediated by ß1AR and ß2AR.

A comprehensive understanding of signalling downstream of GPCRs requires a broad approach to capture novel signalling modalities in addition to established pathways. Here, using an array of sixteen validated BRET-based biosensors, we analyzed the ability of seven different ß-adrenergic ligands to engage five distinct signalling pathways downstream of the ß1-adrenergic receptor (ß1AR). In addition to generating signalling signatures and capturing functional selectivity for the different ligands toward these pathways, we also revealed coupling to signalling pathways that have not previously been ascribed to the ßAR. These include coupling to Gz and G12 pathways. The signalling cascade linking the ß1AR to calcium mobilization was also characterized using a combination of BRET-based biosensors and CRISPR-engineered HEK 293 cells lacking the Gαs subunit or with pharmacological or genetically engineered pathway inhibitors. We show that both Gs and G12 are required for the full calcium response. Our work highlights the power of combining signal profiling with genome editing approaches to capture the full complement of GPCR signalling activities in a given cell type and to probe their underlying mechanisms.

Methods to Monitor the Trafficking of ß-Arrestin/G Protein-Coupled Receptor Complexes Using Enhanced Bystander BRET.

ß-Arrestins are adaptors that regulate the signaling and trafficking of G protein-coupled receptors (GPCRs). Bioluminescence resonance energy transfer (BRET) is a sensitive and versatile method for real-time monitoring of protein-protein interactions and protein kinesis within live cells, such as the recruitment of ß-arrestins to activated receptors at the plasma membrane (PM) and the trafficking of GPCR/ß-arrestin complexes to endosomes. Trafficking of receptor/ß-arrestin complexes can be assessed by BRET through tagging ß-arrestins with the donor luciferase from Renilla reniformis (Rluc) and anchoring the acceptor green fluorescent protein from the same species (rGFP) in distinct cell compartments (e.g., PM or endosomes) to generate highly efficient bystander BRET (referred to as enhanced bystander BRET (EbBRET)) upon re-localization of ß-arrestins to these compartments following receptor activation. Here, we outline the protocol for quantitatively monitoring ß-arrestin recruitment to agonist-activated Angiotensin II type 1 receptor (AT1R) and ß2-adrenergic receptor (ß2AR) at the PM and the trafficking of receptor/ß-arrestin complexes into endosomes using EbBRET-based biosensors.

Functional selectivity profiling of the angiotensin II type 1 receptor using pathway-wide BRET signaling sensors.

G protein-coupled receptors (GPCRs) are important therapeutic targets that exhibit functional selectivity (biased signaling), in which different ligands or receptor variants elicit distinct downstream signaling. Understanding all the signaling events and biases that contribute to both the beneficial and adverse effects of GPCR stimulation by given ligands is important for drug discovery. Here, we report the design, validation, and use of pathway-selective bioluminescence resonance energy transfer (BRET) biosensors that monitor the engagement and activation of signaling effectors downstream of G proteins, including protein kinase C (PKC), phospholipase C (PLC), p63RhoGEF, and Rho. Combined with G protein and ß-arrestin BRET biosensors, our sensors enabled real-time monitoring of GPCR signaling at different levels in downstream pathways in both native and engineered cells. Profiling of the responses to 14 angiotensin II (AngII) type 1 receptor (AT1R) ligands enabled the clustering of compounds into different subfamilies of biased ligands and showed that, in addition to the previously reported functional selectivity between Gαq and ß-arrestin, there are also biases among G protein subtypes. We also demonstrated that biases observed at the receptor and G protein levels propagated to downstream signaling pathways and that these biases could occur through the engagement of different G proteins to activate a common effector. We also used these tools to determine how naturally occurring AT1R variants affected signaling bias. This suite of BRET biosensors provides a useful resource for fingerprinting biased ligands and mutant receptors and for dissecting functional selectivity at various levels of GPCR signaling.

FZD5 is a Gαq-coupled receptor that exhibits the functional hallmarks of prototypical GPCRs.

Frizzleds (FZDs) are a group of seven transmembrane-spanning (7TM) receptors that belong to class F of the G protein-coupled receptor (GPCR) superfamily. FZDs bind WNT proteins to stimulate diverse signaling cascades involved in embryonic development, stem cell regulation, and adult tissue homeostasis. Frizzled 5 (FZD5) is one of the most studied class F GPCRs that promote the functional inactivation of the ß-catenin destruction complex in response to WNTs. However, whether FZDs function as prototypical GPCRs has been heavily debated and, in particular, FZD5 has not been shown to activate heterotrimeric G proteins. Here, we show that FZD5 exhibited a conformational change after the addition of WNT-5A, which is reminiscent of class A and class B GPCR activation. In addition, we performed several live-cell imaging and spectrometric-based approaches, such as dual-color fluorescence recovery after photobleaching (dcFRAP) and resonance energy transfer (RET)-based assays that demonstrated that FZD5 activated Gαq and its downstream effectors upon stimulation with WNT-5A. Together, these findings suggest that FZD5 is a 7TM receptor with a bona fide GPCR activation profile and suggest novel targets for drug discovery in WNT-FZD signaling.

Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D2 Dopamine Receptor.

The dopamine D2 receptor (D2R) is known to elicit effects through activating two major signaling pathways mediated by either G proteins (Gi/o) or ß-arrestins. However, the specific role of each pathway in physiological or therapeutic activities is not known with certainty. One approach to the dissection of these pathways is through the use of drugs that can selectively modulate one pathway vs. the other through a mechanism known as functional selectivity or biased signaling. Our laboratory has previously described a G protein signaling-biased agonist, MLS1547, for the D2R using a variety of in vitro functional assays. To further evaluate the biased signaling activity of this compound, we investigated its ability to promote D2R internalization, a process known to be mediated by ß-arrestin. Using multiple cellular systems and techniques, we found that MLS1547 promotes little D2R internalization, which is consistent with its inability to recruit ß-arrestin. Importantly, we validated these results in primary striatal neurons where the D2R is most highly expressed suggesting that MLS1547 will exhibit biased signaling activity in vivo. In an effort to optimize and further explore structure-activity relationships (SAR) for this scaffold, we conducted an iterative chemistry campaign to synthesize and characterize novel analogs of MLS1547. The resulting analysis confirmed previously described SAR requirements for G protein-biased agonist activity and, importantly, elucidated new structural features that are critical for agonist efficacy and signaling bias of the MLS1547 scaffold. One of the most important determinants for G protein-biased signaling is the interaction of a hydrophobic moiety of the compound with a defined pocket formed by residues within transmembrane five and extracellular loop two of the D2R. These results shed new light on the mechanism of biased signaling of the D2R and may lead to improved functionally-selective molecules.

A new inhibitor of the ß-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling.

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, ß-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of ß-arrestin recruitment to the receptor and ß-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between ß-arrestin and the ß2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/ß-arrestin complexes. This selective ß-arrestin/ß2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical ß2-adrenergic (ß2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect ß-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and ß2AR, supporting the concept of ß-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.

Monitoring G protein-coupled receptor and ß-arrestin trafficking in live cells using enhanced bystander BRET.

Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however, robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and ß-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligand-promoted recruitment or sequestration of RLuc-tagged proteins to, or from, specific cell compartments, as well as sensitive subcellular BRET imaging for protein translocation visualization. These sensors are scalable to high-throughput formats and allow quantitative pharmacological studies of GPCR trafficking in real time, in live cells, revealing ligand-dependent biased trafficking of receptor/ß-arrestin complexes.

Quantifying biased signaling in GPCRs using BRET-based biosensors.

There has been a growing appreciation that G protein-coupled receptor (GPCR) functional selectivity (viz. biased signaling), in particular between G protein- and ß-arrestin-dependent signaling, can be achieved with specific ligands, and that such directed signaling represents a promising avenue for improving drug efficacy and therapy. Thus, for any given GPCRs it is important to define means to pharmacologically characterize and classify drugs for their propensity to bias signaling. Here we describe an experimental protocol and step-by-step approach to assess functional selectivity between Gαq and ß-arrestin-dependent responses using the prototypical angiotensin II (AngII) type 1 receptor (AT1R) expressed in HEK 293 cells. The protocol describes the expression of Bioluminescence Resonance Energy Transfer (BRET) sensors for either Gαq or ß-arrestin with AT1R, and the use of the operational model of pharmacological agonism to quantify ligand bias. Such methods are equally applicable to other GPCRs and their downstream signaling effectors.

ß-Arrestin-mediated Angiotensin II Signaling Controls the Activation of ARF6 Protein and Endocytosis in Migration of Vascular Smooth Muscle Cells.

Angiotensin II (Ang II) is a vasopressive hormone but is also a potent activator of cellular migration. We have previously shown that it can promote the activation of the GTPase ARF6 in a heterologous overexpressing system. The molecular mechanisms by which receptors control the activation of this small G protein remain, however, largely unknown. Furthermore, how ARF6 coordinates the activation of complex cellular responses needs to be further elucidated. In this study, we demonstrate that Ang II receptors engage ß-arrestin, but not Gq, to mediate ARF6 activation in HEK 293 cells. To further confirm the key role of ß-arrestin proteins, we overexpressed ß-arrestin2-(1-320), a dominant negative mutant known to block receptor endocytosis. We show that expression of this truncated construct does not support the activation of the GTPase nor cell migration. Interestingly, ß-arrestin2 can interact with the ARF guanine nucleotide exchange factor ARNO, although the C-terminally lacking mutant does not. We finally examined whether receptor endocytosis controlled ARF6 activation and cell migration. Although the clathrin inhibitor PitStop2 did not impact the ability of Ang II to activate ARF6, cell migration was markedly impaired. To further show that ARF activation regulates key signaling events leading to migration, we also examined MAPK activation. We demonstrate that this signaling axis is relevant in smooth muscle cells of the vasculature. Altogether, our findings show for the first time that Ang II receptor signaling to ß-arrestin regulates ARF6 activation. These proteins together control receptor endocytosis and ultimately cell migration.

The experimental power of FR900359 to study Gq-regulated biological processes.

Despite the discovery of heterotrimeric αßγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq.

Differential regulation of endosomal GPCR/ß-arrestin complexes and trafficking by MAPK.

ß-Arrestins are signaling adaptors that bind to agonist-occupied G protein-coupled receptors (GPCRs) and target them for endocytosis; however, the mechanisms regulating receptor/ß-arrestin complexes and trafficking in endosomes, remain ill defined. Here we show, in live cells, differential dynamic regulation of endosomal bradykinin B2 receptor (B2R) complexes with either ß-arrestin-1 or -2. We find a novel role for MAPK in the B2R/ß-arrestin-2 complex formation, receptor trafficking and signaling mediated by an ERK1/2 regulatory motif in the hinge domain of the rat ß-arrestin-2 (PET(178)P), but not rat ß-arrestin-1 (PER(177)P). While the ERK1/2 regulatory motif is conserved between rat and mouse ß-arrestin-2, it is surprisingly not conserved in human ß-arrestin-2 (PEK(178)P). However, mutation of lysine 178 to threonine is sufficient to confer MAPK sensitivity to the human ß-arrestin-2. Furthermore, substitution for a phosphomimetic residue in both the rat and the human ß-arrestin-2 (T/K178D) significantly stabilizes B2R/ß-arrestin complexes in endosomes, delays receptor recycling to the plasma membrane and maintains intracellular MAPK signaling. Similarly, the endosomal trafficking of ß2-adrenergic, angiotensin II type 1 and vasopressin V2 receptors was altered by the ß-arrestin-2 T178D mutant. Our findings unveil a novel subtype specific mode of MAPK-dependent regulation of ß-arrestins in intracellular trafficking and signaling of GPCRs, and suggest differential endosomal receptor/ß-arrestin-2 signaling roles among species.

G protein-coupled receptor kinase-2 constitutively regulates D2 dopamine receptor expression and signaling independently of receptor phosphorylation.

We investigated the regulatory effects of GRK2 on D(2) dopamine receptor signaling and found that this kinase inhibits both receptor expression and functional signaling in a phosphorylation-independent manner, apparently through different mechanisms. Overexpression of GRK2 was found to suppress receptor expression at the cell surface and enhance agonist-induced internalization, whereas short interfering RNA knockdown of endogenous GRK2 led to an increase in cell surface receptor expression and decreased agonist-mediated endocytosis. These effects were not due to GRK2-mediated phosphorylation of the D(2) receptor as a phosphorylation-null receptor mutant was regulated similarly, and overexpression of a catalytically inactive mutant of GRK2 produced the same effects. The suppression of receptor expression is correlated with constitutive association of GRK2 with the receptor complex as we found that GRK2 and several of its mutants were able to co-immunoprecipitate with the D(2) receptor. Agonist pretreatment did not enhance the ability of GRK2 to co-immunoprecipitate with the receptor. We also found that overexpression of GRK2 attenuated the functional coupling of the D(2) receptor and that this activity required the kinase activity of GRK2 but did not involve receptor phosphorylation, thus suggesting the involvement of an additional GRK2 substrate. Interestingly, we found that the suppression of functional signaling also required the G betagamma binding activity of GRK2 but did not involve the GRK2 N-terminal RH domain. Our results suggest a novel mechanism by which GRK2 negatively regulates G protein-coupled receptor signaling in a manner that is independent of receptor phosphorylation.

G protein-coupled receptor kinase-mediated phosphorylation regulates post-endocytic trafficking of the D2 dopamine receptor.

We investigated the role of G protein-coupled receptor kinase (GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin association, endocytosis, and intracellular trafficking of the D(2) dopamine receptor (DAR). Agonist activation of D(2) DARs results in rapid and sustained receptor phosphorylation that is solely mediated by GRKs. A survey of GRKs revealed that only GRK2 or GRK3 promotes D(2) DAR phosphorylation. Mutational analyses resulted in the identification of eight serine/threonine residues within the third cytoplasmic loop of the receptor that are phosphorylated by GRK2/3. Simultaneous mutation of these eight residues results in a receptor construct, GRK(-), that is completely devoid of agonist-promoted GRK-mediated receptor phosphorylation. We found that both wild-type (WT) and GRK(-) receptors underwent a similar degree of agonist-induced desensitization as assessed using [(35)S]GTPgammaS binding assays. Similarly, both receptor constructs internalized to the same extent in response to agonist treatment. Furthermore, using bioluminescence resonance energy transfer assays to directly assess receptor association with arrestin3, we found no differences between the WT and GRK(-) receptors. Thus, phosphorylation is not required for arrestin-receptor association or agonist-induced desensitization or internalization. In contrast, when we examined recycling of the D(2) DARs to the cell surface, subsequent to agonist-induced endocytosis, the GRK(-) construct exhibited less recycling in comparison with the WT receptor. This impairment appears to be due to a greater propensity of the GRK(-) receptors to down-regulate once internalized. In contrast, if the receptor is highly phosphorylated, then receptor recycling is promoted. These results reveal a novel role for GRK-mediated phosphorylation in regulating the post-endocytic trafficking of a G protein-coupled receptor.