Research Article

Spinophilin Blocks Arrestin Actions in Vitro and in Vivo at G Protein-Coupled Receptors

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Science  25 Jun 2004:
Vol. 304, Issue 5679, pp. 1940-1944
DOI: 10.1126/science.1098274

Abstract

Arrestin regulates almost all G protein–coupled receptor (GPCR)–mediated signaling and trafficking. We report that the multidomain protein, spinophilin, antagonizes these multiple arrestin functions. Through blocking G protein receptor kinase 2 (GRK2) association with receptor-Gβγ complexes, spinophilin reduces arrestin-stabilized receptor phosphorylation, receptor endocytosis, and the acceleration of mitogen-activated protein kinase (MAPK) activity following endocytosis. Spinophilin knockout mice were more sensitive than wild-type mice to sedation elicited by stimulation of α2 adrenergic receptors, whereas arrestin 3 knockout mice were more resistant, indicating that the signal-promoting, rather than the signal-terminating, roles of arrestin are more important for certain response pathways. The reciprocal interactions of GPCRs with spinophilin and arrestin represent a regulatory mechanism for fine-tuning complex receptor-orchestrated cell signaling and responses.

G protein–coupled receptors (GPCRs) are a major target for hormones, neurotransmitters, and cytokines. The signaling and trafficking of these receptors are regulated in multifaceted ways by arrestins (1). Arrestin 1 and 4, visual arrestins for rods and cones, respectively, desensitize rhodopsin signaling (2). Ubiquitously expressed arrestin 2 and 3 (β arrestin 1 and 2) associate with agonist-evoked conformations of GPCRs that are phosphorylated by GRKs to mediate desensitization (3, 4) and endocytosis via clathrin-coated pits (5). The regions of GPCRs that interact with arrestin 2 and 3 are also involved in other GPCR-protein interactions, suggesting that the impact of arrestin on GPCR signaling and trafficking might be regulated by these other GPCR-interacting proteins. Arrestin interacts with the predicted third intracellular (3i) loop of α2-adrenergic receptor (AR) subtypes (68) that also interacts with spinophilin (7). Spinophilin is a ubiquitously expressed protein containing an F-actin–binding domain, a phosphatase 1 (PP1) binding and regulatory domain, a protein-interaction PDZ domain, and C-terminal coiled-coil domains (9, 10), and interacts with at least two subfamilies of GPCRs, the α2AR subtypes (11) and the D2 dopamine receptor (12). However, the functional relevance of these interactions has not been clear.

Two of the α2AR subtypes, the α2AAR and α2BAR, are substrates for GRK2-catalyzed phosphorylation (7, 13) and phosphorylation-dependent arrestin binding. Using these α2AR subtypes as a model system, we demonstrate functional antagonism of multiple arrestin functions in vitro and in vivo by spinophilin. It also appears that the signaling sensitization, rather than signaling desensitization, roles of arrestin are more important for certain response pathways.

Spinophilin competes with GRK2 for binding to α2AR-Gβγ. Arrestin binds to an activated GPCR conformation that is phosphorylated by GRKs (1). Because hormone binding enhances spinophilin interaction with α2AR (11), we examined whether this interaction affects association of GRK2 with agonist-occupied receptors. Epinephrine treatment of transfected simian kidney CosM6 cells expressing either hemagglutinin (HA) epitope–tagged α2AAR (Fig. 1A) or α2BAR (14) resulted in stabilization of receptor-GRK2 interactions. This interaction was blocked when spinophilin was also overexpressed (Fig. 1A), even though the amount of GRK2 protein translocated to the cell membrane was not altered. In contrast, overexpression of arrestin 3 did not affect receptor-GRK2 interaction (Fig. 1A).

Fig. 1.

Spinophilin interaction with the α2AAR competes for agonist-induced GRK2 binding to the receptor and is blocked by pertussis toxin and the GRK2-C tail. (A) Spinophilin competes for GRK2 binding to α2AAR. CosM6 cells cotransfected with HA-α2AAR and the indicated cDNAs were stimulated with α2-agonist (100 μM epinephrine + 1 μM propranolol to block βAR). Bar graphs represent quantitative data from three independent experiments. *P < 0.05, comparing the fold change of GRK2 protein detected in the immunoprecipitated complex with HA-α2AAR after agonist stimulation versus no agonist (the latter defined as control, or 1.0-fold). (B) Spinophilin interaction with α2AAR is blocked by previous incubation with pertussis toxin (PTx) or overexpression of GRK2 C-tail (GRK2-CT). In all studies, Western blot analysis confirmed comparable amounts of HA-α2AAR in immunoprecipitated complexes and comparable amounts of each protein in total lysates for each of the lanes shown.

This hormone-enhanced interaction of spinophilin with the α2AR was nearly eliminated when cells were treated with pertussis toxin (PTx) before agonist stimulation (Fig. 1B), a manipulation that blocks receptor–G protein interactions and receptor-dependent G protein αβγ subunit dissociation. Spinophilin-α2AR interaction was also blocked by expression of the receptor with the GRK2–C terminus (Fig. 1B), which sequesters Gβγ subunits in mammalian cells (15), providing evidence that spinophilin recognizes the complex of α2AR with Gβγ.

Spinophilin attenuates arrestin-stabilized α2AR phosphorylation. Because spinophilin perturbs GRK2 association with the α2AR, we predicted that spinophilin would alter GRK2-catalyzed receptor phosphorylation after agonist stimulation. The dynamic interplay of arrestin and spinophilin in regulating phosphorylation of activated α2AR was revealed either by elimination of spinophilin or arrestin expression by genetic engineering (Fig. 2A) or by heterologous overexpression of arrestin or spinophilin (Fig. 2B). The agonist-evoked increase in detectable HA-α2BAR phosphorylation in wild-type (WT) mouse embryonic fibroblasts (MEFs) was attenuated in arrestin 2/3 null (Arr2,3–/–) MEFs, despite a comparable level of receptor expression (Fig. 2A, left). In contrast, the absence of spinophilin (Sp–/–) resulted in increased agonist-elicited phosphorylation of α2BAR compared with that in WT cells (Fig. 2A, right). We interpret the increased phosphorylation to be the result of unimpeded association of arrestin with the α2AR in Sp–/– cells, thus preventing receptor dephosphorylation by phosphatases, an event that continuously occurs at GPCR (16, 17). Evidence for continuous dephosphorylation of GPCRs is our finding that treatment with okadaic acid, a phosphatase inhibitor, enhanced phosphorylation of α2AR by 80% (14).

Fig. 2.

Spinophilin attenuates arrestin-stabilized α2BAR phosphorylation. (A) The reciprocal effect of arrestin and spinophilin on α2BAR phosphorylation detected in genetically altered MEFs. MEFs with the indicated genotypes were transduced with HA-α2BAR and stimulated with α2-agonist (100 μM epinephrine + 1 μM propranolol) for the indicated times. *P < 0.1; **P < 0.05, comparing the fold change of HA-α2BAR phosphorylation (HA-α2BAR phosphorylation in WT MEFs, with no agonist defined as 1.0-fold); n = 3 experiments. (B) Spinophilin antagonizes the effect of arrestin to stabilize α2BAR phosphorylation. CosM6 cells transfected with HA-α2BAR and GRK2, together with the indicated cDNA(s), were stimulated with α2-agonist. **P < 0.05, comparing the fold change of HA-α2BAR phosphorylation after agonist stimulation versus no agonist (the latter defined as 1.0-fold); n = 3 experiments. Total HA-α2BAR in the immunoprecipitates did not vary appreciably for each of the lanes shown, as confirmed by Western blot analysis.

Heterologous overexpression of arrestin 3 in CosM6 cells [which have relatively low levels of endogenous arrestin 3 expression (18, 19)] enhanced phosphorylation of HA-α2BAR in response to agonist stimulation. However, overexpression of spinophilin reduced arrestin-enhanced α2BAR phosphorylation (Fig. 2B), revealing the reciprocal effect of arrestin and spinophilin on the phosphorylation of activated GPCRs. The effect of spinophilin on the marginally detectable α2BAR phosphorylation in the absence of arrestin overexpression was negligible (Fig. 2B), indicating that spinophilin is principally functioning as an antagonist of arrestin actions, and that this effect requires the presence of arrestin to be detected.

Spinophilin antagonizes arrestin-dependent regulation of α2AR-elicited MAPK activation. Because arrestin preferentially interacts with GRK2-phosphorylated GPCR, attenuation of receptor phosphorylation by spinophilin perturbs not only receptor-arrestin interactions but also subsequent arrestin-dependent GPCR regulation. To explore the effect of spinophilin interactions on α2AR signaling, we examined MAPK activation by endogenous α2AAR expressed in MEFs. Epinephrine activation of the endogenous α2AAR in MEFs is blocked by the α2 antagonist, yohimbine, but is evoked by the α2 agonists, clonidine and UK 14,304 (14). Although GPCRs can activate MAPK by G protein–dependent and –independent pathways (20), blockade of MAPK activation by treatment with PTx (14) reveals that activation of MAPK by endogenous α2AAR in MEFs is mediated by the Gi/Go subtype of heterotrimeric G proteins (2123). Time-course analysis of α2AAR-evoked MAPK activation in Arr2,3–/–, Sp–/–, or WT MEFs (Fig. 3) revealed a longer duration of MAPK activation in Arr2,3–/– MEFs compared with that in WT cells after either epinephrine stimulation of naïve cells or restimulation of cells after 5 min of agonist stimulation and 30 min of wash (Fig. 3A). This finding confirms the role of arrestin in receptor desensitization. In contrast, α2AAR-evoked MAPK stimulation desensitized faster in Sp–/– MEFs compared with the response in WT MEFs (Fig. 3B), consistent with the interpretation that in the absence of spinophilin, enhancement of receptor phosphorylation (Fig. 2A) increases arrestin association with the receptor to terminate its productive coupling to Gi. Recapitulation of these phenotypes in WT MEFs where blockade of arrestins or spinophilin expression was achieved by RNA interference (RNAi) (fig. S1, A and B) minimizes the likelihood that the phenotypes observed in Arr2,3–/– or Sp–/– cells are due to the compensatory changes in these knockout MEFs.

Fig. 3.

Spinophilin is a negative regulator of arrestin-accelerated MAPK activation. MEFs express endogenous α2AAR, as revealed by RT-PCR; the binding capacity of endogenous α2AAR is ∼74.3 ± 10.7 fmol/mg protein, assessed at the dissociation constant (KD) for [3H]Rauwolscine (14). (A) Stimulation and restimulation of endogenous α2AAR-evoked MAPK activation in Arr2,3–/– versus WT MEFs (i.e., MEFs isolated from WT mice with the identical genetic background from which corresponding knockout mice were generated). For stimulation, cells were treated with α2-agonist [100 μM epinephrine + 1 μM propranolol (to block βAR) + 1 μM prazosin (to block α1AR)] for the indicated times. Restimulation with α2-agonist occurred after a 5-min stimulation with α2-agonist and a 30-min wash in Dulbecco's minimum essential medium (DMEM) at 37°C. MAPK activity was assessed by Western blotting for dually phosphorylated MAPK and normalized by Western blotting for total MAPK. Representative enhanced chemiluminesence images are Western blots for phosphorylated MAPK. Data from five independent experiments are summarized in the bar graph. *P < 0.05. (B) Stimulation and restimulation of α2AAR-evoked MAPK activation performed as described in (A) in WT versus Sp–/– MEFs. *P < 0.05; n = 6 experiments. (C) Arrestin is required for agonist-elicited α2AR internalization. HA-α2AAR loss from the surface of MEFs after stimulation with α2-agonist was quantitated by use of an intact cell enzyme-linked immunosorbent assay. Values shown are means ± SEM from 12 experiments. *P < 0.05. (D) Activation of MAPK by endogenous α2AAR in WT versus Sp–/– MEFs incubated in K+-depleted DMEM. At 2 min time point, the MAPK activity values (active/total) in WT and Sp–/– MEFs in K+-depleted DMEM were 0.85 and 0.76, respectively, in the representative experiment. In experiments performed with the same cultures on the same day, values of MAPK activity (active/total) in WT and Sp–/– cells in normal DMEM are 0.88 and 1.38, respectively. The mean values ± range of data from two separate experiments are summarized in the bar graph. (E) GRK phosphorylation–deficient α2AAR mutant evokes sustained MAPK signaling. Stimulation and restimulation of MAPK by WT versus ΔGRK-P α2AAR were examined in human embryonic kidney (HEK) 293 cells expressing endogenous arrestins and spinophilin. The ΔGRK-P α2AAR mutant lacks the LEESSSS sequence in the 3i loop, which represents a substrate site for GRK-catalyzed phosphorylation (7, 13). *P < 0.05, n = 3 experiments.

Arrestin is required for endocytosis of α2AAR (Fig. 3C) and α2BAR (14), whereas spinophilin stabilizes these receptors on the cell surface (24). Our data confirm that endocytosis per se is not essential for α2AAR-mediated MAPK activation (25), because α2AAR stimulated MAPK in Arr2,3–/– cells (Fig. 3A) in the absence of endocytosis (Fig. 3C). Unexpectedly, α2AAR-activated MAPK was more rapidly stimulated in Sp–/– MEFs than in WT MEFs (Fig. 3B). In contrast, the rate of α2AAR-elicited MAPK activation in Arr2,3–/– MEFs was slower than that in WT cells, especially when restimulated after a 5-min stimulation and a 30-min wash (Fig. 3A). In MEFs lacking both arrestin and spinophilin expression, achieved by combined gene knockout and RNA silencing strategies, α2AAR-activated MAPK occurred with a time course parallel to that observed in Arr2,3–/– cells (fig. S1C), indicating that the accelerated MAPK elicited by endogenous α2AAR in Sp–/– MEFs is likely due to facilitated association of arrestin with the receptor.

Endocytosis is a prelude for receptor dephosphorylation and recycling of some GPCRs, including α2AAR and α2BAR (1). Thus, arrestin-dependent acceleration of MAPK stimulation could result from arrestin-dependent endocytosis and subsequent recycling of α2AAR to the surface, replenishing the density of cell surface receptors available for activation. To block endocytosis, we incubated MEFs in a K+-depleted medium that blocks α2AR endocytosis (25). Blockade of endocytosis resulted in prolonged MAPK stimulation at later time points (i.e., 20 min; Fig. 3D), consistent with activation of this enzyme by cell-surface α2AR (25). However, accelerated activation of MAPK observed in Sp–/– cells at 2 min (Fig. 3B) was eliminated when cells were incubated in K+-depleted medium (Fig. 3D).

GRK phosphorylation–deficient α2AAR mutant evokes sustained MAPK signaling. To directly test the influence of the receptor phosphorylation state on the kinetics of MAPK stimulation by α2AR, we compared the time course for MAPK activation by WT α2AAR with that of a mutant α2AAR in which the GRK phosphorylation sites (13) have been deleted (α2AARΔGRK-P). This mutant receptor exhibits enhanced agonist-evoked association with spinophilin (7). Agonist activation of α2AARΔGRK-P resulted in extended duration of MAPK stimulation that mimics the duration of MAPK activation elicited by the WT α2AAR in Arr2,3–/– MEFs (compare Fig. 3, E and A). Thus, attenuating α2AR phosphorylation either by competing for GRK association with the receptor (Fig. 1) or by eliminating the GRK substrate phosphorylation sites (Fig. 3E) results in comparable extension of the duration of receptor-evoked MAPK signaling, presumably due to reduced association of arrestin with the receptor.

Sedation elicited by α2AR is reciprocally regulated in vivo by genetic deletion of arrestin 3 versus spinophilin. To elucidate the in vivo relevance of spinophilin antagonism of arrestin functions, we examined the ability of α2-agonists to evoke sedation mediated by α2AAR (26) in Sp–/– mice, Arr3–/– mice, and corresponding WT mice. Arr3–/– mice were more resistant to UK 14,304-evoked sedation (Fig. 4A, left), indicating that the signal-promoting, rather than signal-terminating, roles of arrestin are more relevant for this particular response. In contrast, sensitivity to α2-agonists was greater in Sp–/– mice (Fig. 4A, right), suggesting that the antagonism of arrestin by spinophilin also occurs in vivo, such that in the absence of spinophilin, receptor-arrestin interactions are facilitated. A1 adenosine receptors, coupled to similar signaling pathways in target cells, also evoked sedation in WT mice, as revealed by their sedative response to the adenosine receptor agonist, R-PIA (Fig. 4B). However, the concentration response curve for R-PIA–evoked sedation was indistinguishable in WT, Sp–/–, or Arr3–/– mice (Fig. 4B). Thus, at least one other receptor pathway remains unaffected in the Arr3–/– and Sp–/– mice, suggesting that the perturbation of α2AAR responses in these mice is not generalized or nonspecific in nature. The differential regulation of spinophilin-arrestin equilibrium on α2AR and A1 adenosine receptor may reflect the discrepancy of spinophilin interaction with these two receptors, or may point to different cellular compartments where these receptors are expressed, because spinophilin is enriched in dendritic spines (10). Hence, the reciprocal regulation of α2AR functions by alternating interactions between spinophilin and arrestin has in vivo relevance.

Fig. 4.

Reciprocal effects of spinophilin and arrestin 3 on α2AR-mediated sedation in vivo. (A) Sedation in response to α2AR-agonists was assessed via rotarod latency after administration of increasing doses of the α2-agonist UK 14,304, as described (26). The EC50 values for sedation in Arr3–/– and corresponding WT littermates (n = 5 for each genotype) are 2.1 and 1.1 mg/kg, respectively, and the EC50 values for sedation in Sp–/– and corresponding WT mice (n = 6 for dose 0.56 mg/kg and n = 11 for the rest doses for each genotype) are 0.4 and 1.2 mg/kg, respectively. *P < 0.01; **P < 0.05. (B) Adenosine A 1R–elicited sedation responses measured with increasing doses of adenosine analog R-PIA. The EC50 values for sedation in Arr3–/– and corresponding WT littermates (n = 5 for each genotype) are 0.86 and 0.77 mg/kg, respectively, and for sedation in Sp–/– and corresponding WT mice (n = 6 for each genotype) are 0.94 and 1.14 mg/kg, respectively.

Discussion. In vitro studies have implicated arrestin in both the termination of and the fostering of distinct GPCR signaling pathways (1). To date, studies in mice lacking arrestin have affirmed its importance in agonist-evoked desensitization pathways (2730). Studies of α2AAR-elicited sedation demonstrate that arrestin's role in enhancing signal activation outweighs its role in deactivating signal transduction in this response pathway (Fig. 4). Furthermore, the data indicate that the multiple functions of arrestin, in vitro and in vivo, are reciprocally regulated by spinophilin in an antagonistic fashion. Consequently, phenotypes observed in Sp–/– mice [reduced long-term depression, resistance to kainate-induced seizures, and neuronal apoptosis (31)] could be due, at least in part, to unimpeded actions of arrestin in modulating these responses.

That spinophilin antagonizes multiple arrestin functions reveals yet another level of complexity of GPCR-orchestrated signaling (Fig. 5). On the basis of findings for the α2A and α2BAR subtypes, spinophilin interaction with activated GPCRs competes for GRK2 binding to the agonist-GPCR-Gβγ complex and thereby blocks receptor phosphorylation. The phosphorylation state of the receptor dictates the relative affinity for arrestin or spinophilin (7, 32). Thus, blockade of GRK2 association with the receptor and subsequent diminished receptor phosphorylation may be directly responsible for diminished arrestin 3 binding to the α2AR and thus antagonism of arrestin's many functions by spinophilin, although direct competition for arrestin interactions by spinophilin binding (7) or spinophilin-dependent binding to other proteins also may contribute to spinophilin antagonism of arrestin functions. The present findings also provide a possible molecular explanation for why arrestin-mediated internalization promotes MAPK activation by GPCR in some systems. Although endocytosis per se is not required for α2AAR-stimulated MAPK (Fig. 3), acceleration of the initial rate of α2AAR-elicited MAPK stimulation under conditions favoring arrestin association with the α2AAR (Sp–/– cells; Fig. 3B) suggests that endocytosis may serve to regenerate agonist-naïve receptor, leading to accelerated or enhanced MAPK activation due to replenishment of activatable receptors (33). The ability of α2AR to activate MAPK in Arr2,3–/– cells indicates that arrestins are not obligatory for MAPK activation by these receptors. Although other GPCR-interacting proteins might be postulated to serve as compensatory scaffolds for MAPK activation in Arr2,3–/– cells, our use of RNAi to eliminate spinophilin expression in Arr2,3–/– MEFs or to reduce arrestin expression in Sp–/– cells (fig. S1C) indicates that spinophilin is not serving as such a postulated compensatory scaffold.

Fig. 5.

Model of GPCR interactions with G proteins, arrestin, and spinophilin, and their functional consequences. Agonist activation of GPCR enhances or stabilizes interactions with G proteins. GRK2 interacts with the GPCR-Gβγ complex and mediates GPCR phosphorylation. Spinophilin competes for GRK2 association with the GPCR-Gβγ complex because activation of the α2AR enhances spinophilin interactions and PTx treatment or coexpression of GRK2-C terminus blocks these interactions. GRK2-catalyzed phosphorylation of the α2A or α2BAR is stabilized by arrestin 3 binding to the receptor, and diminished by increasing the concentration of spinophilin relative to that of arrestin in target cells. The present studies specifically examined arrestin 3, with which α2AR preferentially interacts (8). Arrestin 2 or 3 binding to GPCR enhances desensitization by disrupting coupling to G proteins (1, 35) and enhances endocytosis by facilitating binding to the clathrin-coated pit machinery (1). One outcome of endocytosis is receptor recycling, which facilitates resensitization of receptor responses (19). We postulate that it is this replenishment of recycled, activated α2AAR that contributes to the arrestin-accelerated rate of MAPK signaling observed for the endogenous α2AAR in MEFs (Fig. 3).

Supporting Online Material

www.sciencemag.org/cgi/content/full/304/5679/1940/DC1

Materials and Methods

Fig. S1

References

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