Pyrimidinyl Biphenylureas Act as Allosteric Modulators to Activate Cannabinoid Receptor 1 and Initiate ß-Arrestin-Dependent Responses.

Cannabinoid receptor 1 (CB1) is a G-protein-coupled receptor that is abundant in the central nervous system. It binds several compounds in its orthosteric site, including the endocannabinoids, arachidonoyl ethanolamide (anandamide) and 2-arachidonoyl glycerol, and the plant-derived Δ9-tetrahydrocannabinol, one of the main psychoactive components of marijuana. It primarily couples to Gi/o proteins to inhibit adenylate cyclase activity and typically induces downstream signaling that is Gi-dependent. Since this receptor is implicated in several maladies, such as obesity, pain, and neurodegenerative disorders, there is interest in developing therapeutics that selectively target this receptor. Allosteric modulators of CB1 offer one new approach that has tremendous therapeutic potential. Here, we reveal receptor- and cellular-level properties consistent with receptor activation by a series of pyrimidinyl biphenylureas (LDK1285, LDK1288, LDK1305, and PSNCBAM1), including promoting binding of the agonist CP55940 with positive cooperativity and inhibiting binding of the inverse agonist SR141716A with negative cooperativity, demonstrated via radioligand binding studies. Consistent with these findings, the allosteric modulators induced cellular internalization of the receptor and recruitment of ß-arrestin 2 in human embryonic kidney cell line 293 cells monitored with confocal and total internal reflective fluorescence microscopy, respectively. These allosteric modulators, however, caused G-protein-independent but ß-arrestin 1-dependent phosphorylation of the downstream kinases extracellular signal-regulated kinase 1/2, mitogen-activated protein kinase, and Src, shown by immunoblotting studies. These results are consistent with the involvement of ß-arrestin and suggest that these allosteric modulators induce biased signaling.

Retromer stops beta-arrestin 1-mediated signaling from internalized cannabinoid 2 receptors.

G protein-coupled receptors mediate their complex functions through activation of signaling cascades from receptors localized at the cell surface and endosomal compartments. These signaling pathways are modulated by heterotrimeric G proteins and the scaffold proteins beta-arrestin 1 and 2. However, in contrast to the events occurring at the cell surface, our knowledge of the mechanisms controlling signaling from receptors localized at intracellular compartments is still very limited. Here we sought to investigate the intracellular signaling from cannabinoid 2 receptor (CB2R). First, we show that receptor internalization is required for agonist-induced phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Then we demonstrate that ERK1/2 activation is mediated by beta-arrestin 1 from receptors localized exclusively at Rab4/5 compartments. Finally, we identify the retromer complex as a gatekeeper, terminating beta-arrestin 1-mediated ERK phosphorylation. These findings extend our understanding of the events controlling signaling from endocytosed receptors and identify the retromer as a modulator of beta-arrestin-mediated signaling from CB2R.

The Multiple Waves of Cannabinoid 1 Receptor Signaling.

The cannabinoid 1 receptor (CB1R) is one of the most abundant G protein-coupled receptors (GPCRs) in the central nervous system, with key roles during neurotransmitter release and synaptic plasticity. Upon ligand activation, CB1Rs may signal in three different spatiotemporal waves. The first wave, which is transient (<10 minutes) and initiated by heterotrimeric G proteins, is followed by a second wave (>5 minutes) that is mediated by ß-arrestins. The third and final wave occurs at intracellular compartments and could be elicited by G proteins or ß-arrestins. This complexity presents multiple challenges, including the correct classification of receptor ligands, the identification of the signaling pathways regulated by each wave, and the underlying molecular mechanisms and physiologic impacts of these waves. Simultaneously, it provides new opportunities to harness the therapeutic potential of the cannabinoid system and other GPCRs. Over the last several years, we have significantly expanded our understanding of the mechanisms and pathways downstream from the CB1R. The identification of receptor mutations that can bias signaling to specific pathways and the use of siRNA technology have been key tools to identifying which signaling cascades are controlled by G proteins or ß-arrestins. Here, we review our current knowledge on CB1R signaling, with particular emphasis on the mechanisms and cascades mediated by ß-arrestins downstream from the CB1R.

Mechanisms of Biased ß-Arrestin-Mediated Signaling Downstream from the Cannabinoid 1 Receptor.

Activation of G protein-coupled receptors results in multiple waves of signaling that are mediated by heterotrimeric G proteins and the scaffolding proteins ß-arrestin 1/2. Ligands can elicit full or subsets of cellular responses, a concept defined as ligand bias or functional selectivity. However, our current understanding of ß-arrestin-mediated signaling is still very limited. Here we provide a comprehensive view of ß-arrestin-mediated signaling from the cannabinoid 1 receptor (CB1R). By using a signaling biased receptor, we define the cascades, specific receptor kinases, and molecular mechanism underlying ß-arrestin-mediated signaling: We identify the interaction kinetics of CB1R and ß-arrestin 1 during their endocytic trafficking as directly proportional to its efficacy. Finally, we demonstrate that signaling results in the control of genes clustered around prosurvival and proapoptotic functions among others. Together, these studies constitute a comprehensive description of ß-arrestin-mediated signaling from CB1Rs and suggest modulation of receptor endocytic trafficking as a therapeutic approach to control ß-arrestin-mediated signaling.

Imaging GPCRs trafficking and signaling with total internal reflection fluorescence microscopy in cultured neurons.

Total internal reflection fluorescence (TIRF) microscopy allows probing the cellular events occurring close and at the plasma membrane. Over the last decade, we have seen a significant increase in the number of publications applying TIRF microscopy to unravel some of the fundamental biological questions regarding G protein-coupled receptors (GPCRs) function such as the mechanisms controlling receptor trafficking, quaternary structure, and signaling among others. Most of the published work has been performed in heterologous systems such as HEK293 and CHO cells, where the imaging surface available is higher and smoother when compared with the narrow processes or the smaller cell bodies of neurons. However, some publications have expanded our understanding of these events to primary cell cultures, mostly rat hippocampal and striatal neuronal cultures. Results from these cells provide a bona fide model of the complex events controlling GPCR function in living cells. We believe more work needs to be performed in primary cultures and eventually in intact tissue to complement the knowledge obtained from heterologous cell models. Here, we described a step-by-step protocol to investigate the surface trafficking and signaling from GPCRs in rat hippocampal and striatal primary cultures.

Cannabinoid Receptors in the Central Nervous System: Their Signaling and Roles in Disease.

The identification and cloning of the two major cannabinoid (CB1 and CB2) receptors together with the discovery of their endogenous ligands in the late 80s and early 90s, resulted in a major effort aimed at understanding the mechanisms and physiological roles of the endocannabinoid system (ECS). Due to its expression and localization in the central nervous system (CNS), the CB1 receptor together with its endogenous ligands (endocannabinoids (eCB)) and the enzymes involved in their synthesis and degradation, has been implicated in multiple pathophysiological events ranging from memory deficits to neurodegenerative disorders among others. In this review, we will provide a general overview of the ECS with emphasis on the CB1 receptor in health and disease. We will describe our current understanding of the complex aspects of receptor signaling and trafficking, including the non-canonical signaling pathways such as those mediated by ß-arrestins within the context of functional selectivity and ligand bias. Finally, we will highlight some of the disorders in which CB1 receptors have been implicated. Significant knowledge has been achieved over the last 30 years. However, much more research is still needed to fully understand the complex roles of the ECS, particularly in vivo and to unlock its true potential as a source of therapeutic targets.

Time-Dependent Effects of Ethanol on BK Channel Expression and Trafficking in Hippocampal Neurons.

BACKGROUND: The large conductance Ca(2+) - and voltage-activated K(+) channel (BK) is an important player in molecular and behavioral alcohol tolerance. Trafficking and surface expression of ion channels contribute to the development of addictive behaviors. We have previously reported that internalization of the BK channel is a component of molecular tolerance to ethanol (EtOH). METHODS: Using primary cultures of hippocampal neurons, we combine total internal reflection fluorescence microscopy, electrophysiology, and biochemical techniques to explore how exposure to EtOH affects the expression and subcellular localization of endogenous BK channels over time. RESULTS: Exposure to EtOH changed the expression of endogenous BK channels in a time-dependent manner at the perimembrane area (plasma membrane and/or the area adjacent to it), while total protein levels of BK remain unchanged. These results suggest a redistribution of the channel within the neurons rather than changes in synthesis or degradation rates. Our results showed a temporally nonlinear effect of EtOH on perimembrane expression of BK. First, there was an increase in BK perimembrane expression after 10 minutes of EtOH exposure that remained evident after 3 hours, although not correlated to increases in functional channel expression. In contrast, after 6 hours of EtOH exposure, we observed a significant decrease in both BK perimembrane expression and functional channel expression. Furthermore, after 24 hours of EtOH exposure, perimembrane levels of BK had returned to baseline. CONCLUSIONS: We report a complex time-dependent pattern in the effect of EtOH on BK channel trafficking, including successive increases and decreases in perimembrane expression and a reduction in active BK channels after 3 and 6 hours of EtOH exposure. Possible mechanisms underlying this multiphasic trafficking are discussed. As molecular tolerance necessarily underlies behavioral tolerance, the time-dependent alterations we see at the level of the channel may be relevant to the influence of drinking patterns on the development of behavioral tolerance.

Imaging of kiss-and-run exocytosis of surface receptors in neuronal cultures.

Transmembrane proteins are continuously shuttled from the endosomal compartment to the neuronal plasma membrane by highly regulated and complex trafficking steps. These events are involved in many homeostatic and physiological processes such as neuronal growth, signaling, learning and memory among others. We have previously shown that endosomal exocytosis of the B2 adrenergic receptor (B2AR) and the GluR1-containing AMPA receptor to the neuronal plasma membrane is mediated by two different types of vesicular fusion. A rapid type of exocytosis in which receptors are delivered to the plasma membrane in a single kinetic step, and a persistent mode in which receptors remain clustered at the insertion site for a variable period of time before delivery to the cell surface. Here, by comparing the exocytosis of multiple receptors in dissociated hippocampal and striatal cultures, we show that persistent events are a general mechanism of vesicular delivery. Persistent events were only observed after 10 days in vitro, and their frequency increased with use of the calcium ionophore A23187 and with depolarization induced by KCl. Finally, we determined that vesicles producing persistent events remain at the plasma membrane, closing and reopening their fusion pore for a consecutive release of cargo in a mechanism reminiscent of synaptic kiss-and-run. These results indicate that the delivery of transmembrane receptors to the cell surface can be dynamically regulated by kiss-and-run exocytosis.

Ligand-specific endocytic dwell times control functional selectivity of the cannabinoid receptor 1.

G protein-coupled receptors (GPCRs) are the major transducers of external stimuli and key therapeutic targets in many pathological conditions. When activated by different ligands, one receptor can elicit multiple signalling cascades that are mediated by G proteins or ß-arrestin, a process defined as functional selectivity or ligand bias. However, the dynamic mechanisms underlying ß-arrestin signalling remain unknown. Here by studying the cannabinoid receptor 1 (CB1R), we identify ligand-specific endocytic dwell times, that is, the time during which receptors are clustered into clathrin pits together with ß-arrestins before endocytosis, as the mechanism controlling ß-arrestin signalling. Agonists inducing short endocytic dwell times produce little or no ß-arrestin signalling, whereas those eliciting prolonged dwell times induce robust signalling. Remarkably, extending CB1R dwell times by preventing endocytosis substantially increased ß-arrestin signalling. These studies reveal how receptor activation translates into ß-arrestin signalling and identify a mechanism to control this pathway.

Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing.

Adenosine deaminases that act on RNA are a conserved family of enzymes that catalyze a natural process of site-directed mutagenesis. Biochemically, they convert adenosine to inosine, a nucleotide that is read as guanosine during translation; thus when editing occurs in mRNAs, codons can be recoded and the changes can alter protein function. By removing the endogenous targeting domains from human adenosine deaminase that acts on RNA 2 and replacing them with an antisense RNA oligonucleotide, we have engineered a recombinant enzyme that can be directed to edit anywhere along the RNA registry. Here we demonstrate that this enzyme can efficiently and selectively edit a single adenosine. As proof of principle in vitro, we correct a premature termination codon in mRNAs encoding the cystic fibrosis transmembrane conductance regulator anion channel. In Xenopus oocytes, we show that a genetically encoded version of our editase can correct cystic fibrosis transmembrane conductance regulator mRNA, restore full-length protein, and reestablish functional chloride currents across the plasma membrane. Finally, in a human cell line, we show that a genetically encoded version of our editase and guide RNA can correct a nonfunctional version of enhanced green fluorescent protein, which contains a premature termination codon. This technology should spearhead powerful approaches to correcting a wide variety of genetic mutations and fine-tuning protein function through targeted nucleotide deamination.

Acute inactivation of PSD-95 destabilizes AMPA receptors at hippocampal synapses.

Postsynatptic density protein (PSD-95) is a 95 kDa scaffolding protein that assembles signaling complexes at synapses. Over-expression of PSD-95 in primary hippocampal neurons selectively increases synaptic localization of AMPA receptors; however, mice lacking PSD-95 display grossly normal glutamatergic transmission in hippocampus. To further study the scaffolding role of PSD-95 at excitatory synapses, we generated a recombinant PSD-95-4c containing a tetracysteine motif, which specifically binds a fluorescein derivative and allows for acute and permanent inactivation of PSD-95. Interestingly, acute inactivation of PSD-95 in rat hippocampal cultures rapidly reduced surface AMPA receptor immunostaining, but did not affected NMDA or transferrin receptor localization. Acute photoinactivation of PSD-95 in dissociated neurons causes ∼80% decrease in GluR2 surface staining observed by live-cell microscopy within 15 minutes of PSD-95-4c ablation. These results confirm that PSD-95 stabilizes AMPA receptors at postsynaptic sites and provides insight into the dynamic interplay between PSD-95 and AMPA receptors in live neurons.

Investigating G protein-coupled receptor endocytosis and trafficking by TIR-FM.

G protein-coupled receptors (GPCRs) represent the largest and most versatile family of signaling receptors. Their actions are highly regulated, both under physiological conditions and in response to clinically relevant drugs. A key element in this regulation is control of the number of functional receptors at the cell surface. Major processes that mediate this regulation are vesicular endocytosis and exocytosis of receptors. These trafficking events involve a concerted series of steps, some of which occur on a rapid timescale similar to that of functional signaling itself. Here, we describe and discuss an optical imaging approach, based on evanescent field or total internal reflection-fluorescence microscopy (TIR-FM), to investigate receptor endocytosis and recycling at the level of discrete membrane fission and fusion events. TIR-FM facilitates the study of receptor trafficking events near the plasma membrane with much greater spatial and temporal resolution than afforded by traditional methods. TIR-FM has already provided new insight to GPCR regulation, and we believe that this method has great potential for addressing a variety of questions in GPCR biology.

{Beta}-blocker drugs mediate calcium signaling in native central nervous system neurons by {beta}-arrestin-biased agonism.

G protein-coupled receptors (GPCRs), the largest family of signaling receptors expressed in the CNS, mediate the neuropsychiatric effects of a diverse range of clinically relevant drugs. It is increasingly clear that GPCRs can activate distinct G protein-dependent and -independent transduction pathway(s), and that certain drugs differ in the ability to regulate distinct signaling mechanisms linked to the same receptors. A fundamental question in neuropharmacology is whether such "biased agonism" occurs in physiologically relevant neurons and with endogenous receptors. Here we show that propranolol and carvedilol, two ß-blocker drugs that inhibit ß-adrenergic signaling via heterotrimeric G proteins, function in hippocampal pyramidal neurons as potent and selective activators of an alternate receptor-linked calcium signaling pathway mediated by ß-arrestin-2 and ERK1/2. Our results support the emerging view of ß-arrestin-biased agonism as a significant mechanism of drug action and do so in CNS-derived neurons expressing only native receptors.

Rapid delivery of internalized signaling receptors to the somatodendritic surface by sequence-specific local insertion.

The recycling pathway is a major route for delivering signaling receptors to the somatodendritic plasma membrane. We investigated the cell biological basis for the remarkable selectivity and speed of this process. We focused on the mu-opioid neuropeptide receptor and the beta(2)-adrenergic catecholamine receptor, two seven-transmembrane signaling receptors that traverse the recycling pathway efficiently after ligand-induced endocytosis and localize at steady state throughout the postsynaptic surface. Rapid recycling of each receptor in dissociated neuronal cultures was mediated by a receptor-specific cytoplasmic sorting sequence. Total internal reflection fluorescence microscopy imaging revealed that both sequences drive recycling via discrete vesicular fusion events in the cell body and dendritic shaft. Both sequences promoted recycling via "transient"-type events characterized by nearly immediate lateral spread of receptors after vesicular insertion resembling receptor insertion events observed previously in non-neural cells. The sequences differed in their abilities to produce distinct "persistent"-type events at which inserted receptors lingered for a variable time period before lateral spread. Both types of insertion event generated a uniform distribution of receptors in the somatodendritic plasma membrane when imaged over a 1 min interval, but persistent events uniquely generated a punctate surface distribution over a 10 s interval. These results establish sequence-directed recycling of signaling receptors in CNS neurons and show that this mechanism has the ability to generate receptor-specific patterns of local surface distribution on a timescale overlapping that of rapid physiological signaling.

Cargo-mediated regulation of a rapid Rab4-dependent recycling pathway.

Membrane trafficking is well known to regulate receptor-mediated signaling processes, but less is known about whether signaling receptors conversely regulate the membrane trafficking machinery. We investigated this question by focusing on the beta-2 adrenergic receptor (B2AR), a G protein-coupled receptor whose cellular signaling activity is controlled by ligand-induced endocytosis followed by recycling. We used total internal reflection fluorescence microscopy (TIR-FM) and tagging with a pH-sensitive GFP variant to image discrete membrane trafficking events mediating B2AR endo- and exocytosis. Within several minutes after initiating rapid endocytosis of B2ARs by the adrenergic agonist isoproterenol, we observed bright "puffs" of locally increased surface fluorescence intensity representing discrete Rab4-dependent recycling events. These events reached a constant frequency in the continuous presence of isoproterenol, and agonist removal produced a rapid (observed within 1 min) and pronounced (approximately twofold) increase in recycling event frequency. This regulation required receptor signaling via the cAMP-dependent protein kinase (PKA) and a specific PKA consensus site located in the carboxyl-terminal cytoplasmic tail of the B2AR itself. B2AR-mediated regulation was not restricted to this membrane cargo, however, as transferrin receptors packaged in the same population of recycling vesicles were similarly affected. In contrast, net recycling measured over a longer time interval (10 to 30 min) was not detectably regulated by B2AR signaling. These results identify rapid regulation of a specific recycling pathway by a signaling receptor cargo.

Real-time imaging of discrete exocytic events mediating surface delivery of AMPA receptors.

We directly resolved discrete exocytic fusion events mediating insertion of AMPA-type glutamate receptors (AMPARs) to the somatodendritic surface of rat hippocampal pyramidal neurons, in slice and dissociated cultures, using protein tagging with a pH-sensitive GFP (green fluorescent protein) variant and rapid (10 frames/s) fluorescence microscopy. AMPAR-containing exocytic events occurred under basal culture conditions in both the cell body and dendrites; potentiating chemical stimuli produced an NMDA receptor-dependent increase in the frequency of individual exocytic events. The number of AMPARs inserted per exocytic event, estimated using single-molecule analysis, was quite uniform but individual events differed significantly in kinetic properties affecting the subsequent surface distribution of receptors. "Transient" events, from which AMPARs dispersed laterally immediately after surface insertion, generated a pronounced but short-lived (dissipating within approximately 1 s) increase in surface AMPAR fluorescence extending locally (2-5 microm) from the site of exocytosis. "Persistent" events, from which inserted AMPARs dispersed slowly (typically over 5-10 s), affected local surface receptor concentration to a much smaller degree. Both modes of exocytic insertion occurred throughout the dendritic shaft, but remarkably, neither mode of insertion was observed directly into synaptic spines. AMPARs entered spines preferentially from transient events occurring in the adjoining dendritic shaft, driven apparently by mass action and short-range lateral diffusion, and locally delivered AMPARs remained mostly in the mobile fraction. These results suggest a highly dynamic mechanism for both constitutive and activity-dependent surface delivery of AMPARs, mediated by kinetically distinct exocytic modes that differ in propensity to drive lateral entry of receptors to nearby synapses.

Endocytosis of neurotransmitter receptors: location matters.

Endocytosis of excitatory glutamate receptors from the postsynaptic plasma membrane plays a fundamental role in synaptic function and plasticity. In a recent study published in Neuron, Lu et al. (2007) describe protein interactions that link zones of receptor endocytosis directly to the postsynaptic scaffold and propose that local trafficking of receptors facilitated by these endocytic zones is required to maintain synaptic responsiveness.

Distinct modes of regulated receptor insertion to the somatodendritic plasma membrane.

Many neural signaling receptors are regulated by endocytosis, but little is known about receptor insertion into the plasma membrane. Time-lapse imaging of the beta2 adrenergic receptor expressed in cultured rat hippocampal neurons, using pH-sensitive green fluorescent protein tagging and total internal reflection fluorescence microscopy, resolved distinct vesicular fusion events mediating receptor insertion into the somatodendritic plasma membrane. A 'transient' insertion mode resulted in rapid lateral dispersion of receptors immediately after insertion. A 'persistent' insertion mode resulted in the retention of inserted receptors in surface-accessible domains, which were relatively immobile for a prolonged 'wait' period before dispersing laterally. Distinct insertion modes were oppositely regulated by receptor activation and by mechanisms differing in their dependence on the signaling effector cyclic AMP-dependent protein kinase. These results reveal a new mechanism for homeostatic regulation of postsynaptic signaling and a 'kiss-and-wait' mode of regulated membrane protein insertion in neurons.

Evidence for tryptophan residues in the cation transport path of the Na(+),K(+)-ATPase.

A family of aryl isothiouronium derivatives was designed as probes for cation binding sites of Na(+),K(+)-ATPase. Previous work showed that 1-bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU) acts as a competitive blocker of Na(+) or K(+) occlusion. In addition to a high-affinity cytoplasmic site (K(D) < 1 microM), a low-affinity site (K(D) approximately 10 microM) was detected, presumably extracellular. Here we describe properties of Br-TITU as a blocker at the extracellular surface. In human red blood cells Br-TITU inhibits ouabain-sensitive Na(+) transport (K(D) approximately 30 microM) in a manner antagonistic with respect to extracellular Na(+). In addition, Br-TITU impairs K(+)-stimulated dephosphorylation and Rb(+) occlusion from phosphorylated enzyme of renal Na(+),K(+)-ATPase, consistent with binding to an extracellular site. Incubation of renal Na(+),K(+)-ATPase with Br-TITU at pH 9 irreversibly inactivates Na(+),K(+)-ATPase activity and Rb(+) occlusion. Rb(+) or Na(+) ions protect. Preincubation of Br-TITU with red cells in a K(+)-free medium at pH 9 irreversibly inactivates ouabain-sensitive (22)Na(+) efflux, showing that inactivation occurs at an extracellular site. K(+), Cs(+), and Li(+) ions protect against this effect, but the apparent affinity for K(+), Cs(+), or Li(+) is similar (K(D) approximately 5 mM) despite their different affinities for external activation of the Na(+) pump. Br-TITU quenches tryptophan fluorescence of renal Na(+),K(+)-ATPase or of digested "19 kDa membranes". After incubation at pH 9 irreversible loss of tryptophan fluorescence is observed and Rb(+) or Na(+) ions protect. The Br-TITU appears to interact strongly with tryptophan residue(s) within the lipid or at the extracellular membrane-water interface and interfere with cation occlusion and Na(+),K(+)-ATPase activity.

Relevance of dopamine signals anchoring dynamin-2 to the plasma membrane during Na+,K+-ATPase endocytosis.

Clathrin-dependent endocytosis of Na(+),K(+)-ATPase in response to dopamine regulates its catalytic activity in intact cells. Because fission of clathrin-coated pits requires dynamin, we examined the mechanisms by which dopamine receptor signals promote dynamin-2 recruitment and assembly at the site of Na(+),K(+)-ATPase endocytosis. Western blotting revealed that dopamine increased the association of dynamin-2 with the plasma membrane and with phosphatidylinositol 3-kinase. Dopamine inhibited Na(+),K(+)-ATPase activity in OK cells and in those overexpressing wild type dynamin-2 but not in cells expressing a dominant-negative mutant. Dephosphorylation of dynamin is important for its assembly. Dopamine increased protein phosphatase 2A activity and dephosphorylated dynamin-2. In cells expressing a dominant-negative mutant of protein phosphatase 2A, dopamine failed to dephosphorylate dynamin-2 and to reduce Na(+),K(+)-ATPase activity. Dynamin-2 is phosphorylated at Ser(848), and expression of the S848A mutant significantly blocked the inhibitory effect of dopamine. These results demonstrate a distinct signaling network originating from the dopamine receptor that regulates the state of dynamin-2 phosphorylation and that promotes its location (by interaction with phosphatidylinositol 3-kinase) at the site of Na(+),K(+)-ATPase endocytosis.