A generic approach for the purification of signaling complexes that specifically interact with the carboxyl-terminal domain of G protein-coupled receptors

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are major drug targets. Recent progress has shown that GPCRs are part of large protein complexes that regulate their activity. We present here a generic approach for identification of these complexes that is based on the use of receptor subdomains and that overcomes the limitations of currently used genetics and proteomics approaches. Our approach consists of a carefully balanced combination of chemically synthesized His6-tagged baits, immobilized metal affinity chromatography, one- and two-dimensional gel electrophoresis separation and mass spectrometric identification. The carboxyl-terminal tails (C-tails) of the human MT1 and MT2 melatonin receptors, two class A GPCRs, were used as models to purify protein complexes from mouse brain lysates. We identified 32 proteins that interacted with the C-tail of MT1, 14 proteins that interacted with the C-tail of MT2, and eight proteins that interacted with both C-tails. Several randomly selected proteins were validated by Western blotting, and the functional relevance of our data was further confirmed by showing the interaction between the full-length MT1 and the regulator of G protein signaling Z1 in transfected HEK 293 cells and native tissue. Taken together, we have established an integrated and generic purification strategy for the identification of high quality and functionally relevant GPCR-associated protein complexes that significantly widens the repertoire of available techniques.     

Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein-coupled receptors delineate two major classes of receptors

Visual arrestin, betaarrestin1, and betaarrestin2 comprise a family of intracellular proteins that desensitize G protein-coupled receptors (GPCRs). In addition, betaarrestin1 and betaarrestin2 target desensitized receptors to clathrin-coated pits for endocytosis. Whether arrestins differ in their ability to interact with GPCRs in cells is not known. In this study, we visualize the interaction of arrestin family members with GPCRs in real time and in live cells using green fluorescent protein-tagged arrestins. In the absence of agonist, visual arrestin and betaarrestin1 were found in both the cytoplasm and nucleus of HEK-293 cells, whereas betaarrestin2 was found only in the cytoplasm. Analysis of agonist-mediated arrestin translocation to multiple GPCRs identified two major classes of receptors. Class A receptors (beta2 adrenergic receptor, mu opioid receptor, endothelin type A receptor, dopamine D1A receptor, and alpha1b adrenergic receptor) bound betaarrestin2 with higher affinity than betaarrestin1 and did not interact with visual arrestin. In contrast, class B receptors (angiotensin II type 1A receptor, neurotensin receptor 1, vasopressin V2 receptor, thyrotropin-releasing hormone receptor, and substance P receptor) bound both betaarrestin isoforms with similar high affinities and also interacted with visual arrestin. Switching the carboxyl-terminal tails of class A and class B receptors completely reversed the affinity of each receptor for the visual and non-visual arrestins. In addition, exchanging the betaarrestin1 and betaarrestin2 carboxyl termini reversed their extent of binding to class A receptors as well as their subcellular distribution. These results reveal for the first time marked differences in the ability of arrestin family members to bind GPCRs at the plasma membrane. Moreover, they show that visual arrestin can interact in cells with GPCRs other than rhodopsin. These findings suggest that GPCR signaling may be differentially regulated depending on the cellular complement of arrestin isoforms and the ability of arrestins to interact with other cellular proteins.     

A proteomic approach based on peptide affinity chromatography, 2-dimensional electrophoresis and mass spectrometry to identify multiprotein complexes interacting with membrane-bound receptors

There is accumulating evidence that membrane-bound receptors interact with many intracellular proteins. Multiprotein complexes associated with ionotropic receptors have been extensively characterized, but the identification of proteins interacting with G protein-coupled receptors (GPCRs) has so far only been achieved in a piecemeal fashion, focusing on one or two protein species. We describe a method based on peptide affinity chromatography, two-dimensional electrophoresis, mass spectrometry and immunoblotting to identify the components of multiprotein complexes interacting directly or indirectly with intracellular domains of GPCRs or, more generally, any other membrane-bound receptor. Using this global approach, we have characterized multiprotein complexes that bind to the carboxy-terminal tail of the 5-hydroxytryptamine type 2C receptor and are important for its subcellular localization in CNS cells (Bécamel et al., EMBO J., 21(10): 2332, 2002). Published: December 9, 2002     

Identification and characterization of novel nicotinic receptor-associated proteins in Caenorhabditis elegans

Nicotinic acetylcholine receptors (nAChRs) mediate fast excitatory neurotransmission in neurons and muscles. To identify nAChR accessory proteins, which may regulate their expression or function, we performed tandem affinity purification of the levamisole-sensitive nAChR from Caenorhabditis elegans, mass spectrometry of associated components, and RNAi-based screening for effects on in vivo nicotine sensitivity. Among the proteins identified was the calcineurin A subunit TAX-6, which appeared to function as a negative regulator of nAChR activity. We also identified five proteins not previously linked to nAChR function, whose inactivation conferred nicotine resistance, implicating them as positive regulators of nAChR activity. Of these, the copine NRA-1 colocalized with the levamisole receptor at neuronal and muscle plasma membranes, and, when mutated, caused reduced synaptic nAChR expression. Loss of SOC-1, which acts in receptor tyrosine kinase (RTK) signaling, also reduced synaptic levamisole receptor levels, as did mutations in the fibroblast growth factor receptor EGL-15, and another RTK, CAM-1. Thus, tandem affinity purification is a viable approach to identify novel proteins regulating neurotransmitter receptor activity or expression in model systems like C. elegans.     

Identification of a novel family of G protein-coupled receptor associated sorting proteins

During the past few years several new interacting partners for G protein-coupled receptors (GPCRs) have been discovered, suggesting that the activity of these receptors is more complex than previously anticipated. Recently, candidate G protein-coupled receptor associated sorting protein (GASP-1) has been identified as a novel interacting partner for the delta opioid receptor and has been proposed to determine the degradative fate of this receptor. We show here that GASP-1 associates in vitro with other opioid receptors and that the interaction domain in these receptors is restricted to a small portion of the carboxyl-terminal tail, corresponding to helix 8 in the three-dimensional structure of rhodopsin. In addition, we show that GASP-1 interacts with COOH-terminus of several other GPCRs from subfamilies A and B and that two conserved residues within the putative helix 8 of these receptors are critical for the interaction with GASP-1. In situ hybridization and northern blot analysis indicate that GASP-1 mRNA is mainly distributed throughout the central nervous system, consistent with a potential interaction with numerous GPCRs in vivo. Finally, we show that GASP-1 is a member of a novel family comprising at least 10 members, whose genes are clustered on chromosome X. Another member of the family, GASP-2, also interacts with the carboxyl-terminal tail of several GPCRs. Therefore, GASP proteins may represent an important protein family regulating GPCR physiology.     

The 'magic tail' of G protein-coupled receptors: an anchorage for functional protein networks

All cell types express a great variety of G protein-coupled receptors (GPCRs) that are coupled to only a limited set of G proteins. This disposition favors cross-talk between transduction pathways. However, GPCRs are organized into functional units. They promote specificity and thus avoid unsuitable cross-talk. New methodologies (mostly yeast two-hybrid screens and proteomics) have been used to discover more than 50 GPCR-associated proteins that are involved in building these units. In addition, these protein networks participate in the trafficking, targeting, signaling, fine-tuning and allosteric regulation of GPCRs. To date, proteins that interact with the GPCR C-terminus are the most abundant and are the focus of this review.     

Association of beta-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization.

Resensitization of G protein-coupled receptors (GPCRs) following agonist-mediated desensitization is a necessary step for maintaining physiological responsiveness. However, the molecular mechanisms governing the nature of GPCR resensitization are poorly understood. Here, we examine the role of beta-arrestin in the resensitization of the beta(2) adrenergic receptor (beta(2)AR), known to recycle and resensitize rapidly, and the vasopressin V2 receptor (V2R), known to recycle and resensitize slowly. Upon agonist activation, both receptors recruit beta-arrestin to the plasma membrane and internalize in a beta-arrestin- and clathrin-dependent manner. However, whereas beta-arrestin dissociates from the beta(2)AR at the plasma membrane, it internalizes with the V2R into endosomes. The differential trafficking of beta-arrestin and the ability of these two receptors to dephosphorylate, recycle, and resensitize is completely reversed when the carboxyl-terminal tails of these two receptors are switched. Moreover, the ability of beta-arrestin to remain associated with desensitized GPCRs during clathrin-mediated endocytosis is mediated by a specific cluster of phosphorylated serine residues in the receptor carboxyl-terminal tail. These results demonstrate that the interaction of beta-arrestin with a specific motif in the GPCR carboxyl-terminal tail dictates the rate of receptor dephosphorylation, recycling, and resensitization, and thus provide direct evidence for a novel mechanism by which beta-arrestins regulate the reestablishment of GPCR responsiveness.     

Changes in G protein-coupled receptor sorting protein affinity regulate postendocytic targeting of G protein-coupled receptors

After activation, most G protein-coupled receptors (GPCRs) are regulated by a cascade of events involving desensitization and endocytosis. Internalized receptors can then be recycled to the plasma membrane, retained in an endosomal compartment, or targeted for degradation. The GPCR-associated sorting protein, GASP, has been shown to preferentially sort a number of native GPCRs to the lysosome for degradation after endocytosis. Here we show that a mutant beta(2) adrenergic receptor and a mutant mu opioid receptor that have previously been described as lacking "recycling signals" due to mutations in their C termini in fact bind to GASP and are targeted for degradation. We also show that a mutant dopamine D1 receptor, which has likewise been described as lacking a recycling signal, does not bind to GASP and is therefore not targeted for degradation. Together, these results indicate that alteration of receptors in their C termini can expose determinants with affinity for GASP binding and consequently target receptors for degradation.     

Identification of proteins that associate with integrin α2 by proteomic analysis in human fibrosarcoma HT-1080 cells

Integrins are adhesion receptors for components of the extracellular matrix (ECMs) that regulate multiple cellular functions, such as migration, invasion, proliferation, and survival by mediating bidirectional signal transmission. Even though many proteins have been reported to associate with integrins both on and in cells, systemic analyses of the adhesome have not been carried out. In previous studies, we identified proteins associating with a membrane-type protease, MT1-MMP, using nano-flow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) of associated proteins prepared by optimized conditions for cell lysis and purification. Since integrins were identified as MT1-MMP-associated proteins, we next applied this method to analyze integrin-associated proteins. In this study, we expressed integrin α2 fused at the C terminus to a FLAG peptide in HT1080 cells. Cells stably expressing the chimeric protein were lysed with 1% Brij-98 and affinity purified using anti-FLAG antibody. Integrin β1 co-purified with integrin α2 confirming the specificity of the purification procedure. Analysis of the purified mixture by nano-LC/MS/MS identified 70 proteins. Nineteen of these were membrane proteins, including adhesion proteins, receptors, transporters, proteinases, and ion-channel receptors, and the balance were cytoplasmic. Interestingly, eight of the proteins had previously been shown to associate with MT1-MMP. We believe the present study provides a platform to facilitate the study of the mechanisms of cell adhesion, migration, and invasion.     

Functional capabilities of an N-formyl peptide receptor-G(alpha)(i)(2) fusion protein: assemblies with G proteins and arrestins

G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.     

The orphan GPR50 receptor specifically inhibits MT1 melatonin receptor function through heterodimerization

One-third of the approximately 400 nonodorant G protein-coupled receptors (GPCRs) are still orphans. Although a considerable number of these receptors are likely to transduce cellular signals in response to ligands that remain to be identified, they may also have ligand-independent functions. Several members of the GPCR family have been shown to modulate the function of other receptors through heterodimerization. We show that GPR50, an orphan GPCR, heterodimerizes constitutively and specifically with MT(1) and MT(2) melatonin receptors, using biochemical and biophysical approaches in intact cells. Whereas the association between GPR50 and MT(2) did not modify MT(2) function, GPR50 abolished high-affinity agonist binding and G protein coupling to the MT(1) protomer engaged in the heterodimer. Deletion of the large C-terminal tail of GPR50 suppressed the inhibitory effect of GPR50 on MT(1) without affecting heterodimerization, indicating that this domain regulates the interaction of regulatory proteins to MT(1). Pairing orphan GPCRs to potential heterodimerization partners might be of clinical importance and may become a general strategy to better understand the function of orphan GPCRs.     

Receptor/beta-arrestin complex formation and the differential trafficking and resensitization of beta2-adrenergic and angiotensin II type 1A receptors

Beta-arrestins target G protein-coupled receptors (GPCRs) for endocytosis via clathrin-coated vesicles. Beta-arrestins also become detectable on endocytic vesicles in response to angiotensin II type 1A receptor (AT1AR), but not beta2-adrenergic receptor (beta2AR), activation. The carboxyl-terminal tails of these receptors contribute directly to this phenotype, since a beta2AR bearing the AT1AR tail acquired the capacity to stimulate beta-arrestin redistribution to endosomes, whereas this property was lost for an AT1AR bearing the beta2AR tail. Using beta2AR/AT1AR chimeras, we tested whether the beta2AR and AT1AR carboxyl-terminal tails, in part via their association with beta-arrestins, might regulate differences in the intracellular trafficking and resensitization patterns of these receptors. In the present study, we find that beta-arrestin formed a stable complex with the AT1AR tail in endocytic vesicles and that the internalization of this complex was dynamin dependent. Internalization of the beta2AR chimera bearing the AT1AR tail was observed in the absence of agonist and was inhibited by a dominant-negative beta-arrestin1 mutant. Agonist-independent AT1AR internalization was also observed after beta-arrestin2 overexpression. After internalization, the beta2AR, but not the AT1AR, was dephosphorylated and recycled back to the cell surface. However, the AT1AR tail prevented beta2AR dephosphorylation and recycling. In contrast, although the beta2AR-tail promoted AT1AR recycling, the chimeric receptor remained both phosphorylated and desensitized, suggesting that receptor dephosphorylation is not a property common to all receptors. In summary, we show that the carboxyl-terminal tails of GPCRs not only contribute to regulating the patterns of receptor desensitization, but also modulate receptor intracellular trafficking and resensitization patterns.     

Molecular determinants underlying the formation of stable intracellular G protein-coupled receptor-beta-arrestin complexes after receptor endocytosis*

beta-Arrestins bind agonist-activated G protein-coupled receptors (GPCRs) and mediate their desensitization and internalization. Although beta-arrestins dissociate from some receptors at the plasma membrane, such as the beta2 adrenergic receptor, they remain associated with other GPCRs and internalize with them into endocytic vesicles. Formation of stable receptor-beta-arrestin complexes that persist inside the cell impedes receptor resensitization, and the aberrant formation of these complexes may play a role in GPCR-based diseases (Barak, L. S., Oakley, R. H., Laporte, S. A., and Caron, M. G. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 93-98). Here, we investigate the molecular determinants responsible for sustained receptor/beta-arrestin interactions. We show in real time and in live human embryonic kidney (HEK-293) cells that a beta-arrestin-2-green fluorescent protein conjugate internalizes into endocytic vesicles with agonist-activated neurotensin-1 receptor, oxytocin receptor, angiotensin II type 1A receptor, and substance P receptor. Using receptor mutagenesis, we demonstrate that the ability of beta-arrestin to remain associated with these receptors is mediated by specific clusters of serine and threonine residues located in the receptor carboxyl-terminal tail. These clusters are remarkably conserved in their position within the carboxyl-terminal domain and serve as primary sites of agonist-dependent receptor phosphorylation. In addition, we identify a beta-arrestin mutant with enhanced affinity for the agonist-activated beta2-adrenergic receptor that traffics into endocytic vesicles with receptors that lack serine/threonine clusters and normally dissociate from wild-type beta-arrestin at the plasma membrane. By identifying receptor and beta-arrestin residues critical for the formation of stable receptor-beta-arrestin complexes, these studies provide novel targets for regulating GPCR responsiveness and treating diseases resulting from abnormal GPCR/beta-arrestin interactions.     

A novel, generic and effective method for the rapid purification of G protein-coupled receptors

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. Structure determination of G protein-coupled receptors and other applications require milligram quantities of purified receptor proteins on a regular basis. Recombinant GPCRs fused to a heterologous biotinylation domain were produced in the yeast Pichia pastoris. We describe an efficient method for their rapid purification that relies on the capture of these receptors with streptavidin immobilized on agarose beads, and their subsequent release by enzymatic digestion with TEV protease. This method has been applied to several GPCRs belonging to the class A rhodopsin subfamily, leading to high yields of purified proteins; it represents a method of choice for biochemical and biophysical studies when large quantities of purified GPCRs are needed.     

The PDZ protein mupp1 promotes Gi coupling and signaling of the Mt1 melatonin receptor

Intracellular signaling events are often organized around PDZ (PSD-95/Drosophila Disc large/ZO-1 homology) domain-containing scaffolding proteins. The ubiquitously expressed multi-PDZ protein MUPP1, which is composed of 13 PDZ domains, has been shown to interact with multiple viral and cellular proteins and to play important roles in receptor targeting and trafficking. In this study, we show that MUPP1 binds to the G protein-coupled MT(1) melatonin receptor and directly regulates its G(i)-dependent signal transduction. Structural determinants involved in this interaction are the PDZ10 domain of MUPP1 and the valine of the canonical class III PDZ domain binding motif DSV of the MT(1) carboxyl terminus. This high affinity interaction (K(d) approximately 4 nm), which is independent of MT(1) activation, occurs in the ovine pars tuberalis of the pituitary expressing both proteins endogenously. Although the disruption of the MT(1)/MUPP1 interaction has no effect on the subcellular localization, trafficking, or degradation of MT(1), it destabilizes the interaction between MT(1) and G(i) and abolishes G(i)-mediated signaling of MT(1). Our findings highlight a previously unappreciated role of PDZ proteins in promoting G protein coupling to receptors.     

Common structural requirements for heptahelical domain function in class A and class C G protein-coupled receptors

G protein-coupled receptors (GPCRs) are key players in cell communication. Several classes of such receptors have been identified. Although all GPCRs possess a heptahelical domain directly activating G proteins, important structural and sequence differences within receptors from different classes suggested distinct activation mechanisms. Here we show that highly conserved charged residues likely involved in an interaction network between transmembrane domains (TM) 3 and 6 at the cytoplasmic side of class C GPCRs are critical for activation of the gamma-aminobutyric acid type B receptor. Indeed, the loss of function resulting from the mutation of the conserved lysine residue into aspartate or glutamate in the TM3 of gamma-aminobutyric acid type B(2) can be partly rescued by mutating the conserved acidic residue of TM6 into either lysine or arginine. In addition, mutation of the conserved lysine into an acidic residue leads to a nonfunctional receptor that displays a high agonist affinity. This is reminiscent of a similar ionic network that constitutes a lock stabilizing the inactive state of many class A rhodopsin-like GPCRs. These data reveal that despite their original structure, class C GPCRs share with class A receptors at least some common structural feature controlling G protein activation.     

Feedback inhibition of G protein-coupled receptor kinase 2 (GRK2) activity by extracellular signal-regulated kinases

G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and beta-arrestin binding uncouple G protein-coupled receptors (GPCRs) from their respective G proteins and initiates the process of receptor internalization. In the case of the beta(2)-adrenergic receptor and lysophosphatidic acid receptor, these processes can lead to ERK activation. Here we identify a novel mechanism whereby the activity of GRK2 is regulated by feedback inhibition. GRK2 is demonstrated to be a phosphoprotein in cells. Mass spectrometry and mutational analysis localize the site of phosphorylation on GRK2 to a carboxyl-terminal serine residue (Ser(670)). Phosphorylation at Ser(670) impairs the ability of GRK2 to phosphorylate both soluble and membrane-incorporated receptor substrates and dramatically attenuates Gbetagamma-mediated activation of this enzyme. Ser(670) is located in a peptide sequence that conforms to an ERK consensus phosphorylation sequence, and in vitro, in the presence of heparin, ERK1 phosphorylates GRK2. Inhibition of ERK activity in HEK293 cells potentiates GRK2 activity, whereas, conversely, ERK activation inhibits GRK2 activity. The discovery that ERK phosphorylates and inactivates GRK2 suggests that ERK participates in a feedback regulatory loop. By negatively regulating GRK-mediated receptor phosphorylation, beta-arrestin-mediated processes such as Src recruitment and clathrin-mediated internalization, which are required for GPCR-mediated ERK activation, are inhibited, thus dampening further ERK activation.     

The G protein G alpha(13) is required for growth factor-induced cell migration

Heterotrimeric G proteins are critical cellular signal transducers. They are known to directly relay signals from seven-transmembrane G protein-coupled receptors (GPCRs) to downstream effectors. On the other hand, receptor tyrosine kinases (RTKs), a different family of membrane receptors, signal through docking sites in their carboxy-terminal tails created by autophosphorylated tyrosine residues. Here we show that a heterotrimeric G protein, G alpha(13), is essential for RTK-induced migration of mouse fibroblast and endothelial cells. G alpha(13) activity in cell migration is retained in a C-terminal mutant that is defective in GPCR coupling, suggesting that the migration function is independent of GPCR signaling. Thus, G alpha(13) appears to be a critical signal transducer for RTKs as well as GPCRs. This broader role of G alpha(13) in cell migration initiated by two types of receptors could provide a molecular basis for the vascular system defects exhibited by G alpha(13) knockout mice.