Huntington disease is caused by a polyglutamine expansion in the huntingtin protein (Htt) and is associated with excitotoxic death of striatal neurons. Group I metabotropic glutamate receptors (mGluRs) that are coupled to inositol 1,4,5-triphosphate formation and the release of intracellular Ca 2؉ stores play an important role in regulating neuronal function. We show here that mGluRs interact with the Htt-binding protein optineurin that is also linked to normal pressure open angled glaucoma and, when expressed in HEK 293 cells, optineurin functions to antagonize agonist-stimulated mGluR1a signaling. We find that Htt is co-precipitated with mGluR1a and that mutant Htt functions to facilitate optineurinmediated attenuation of mGluR1a signaling. In striatal cell lines derived from Htt Q111/Q111 mutant knock-in mice mGluR5-stimulated inositol phosphate formation is also severely impaired when compared with striatal cells derived from Htt Q7/Q7 knock-in mice. In addition, we show that a missense single nucleotide polymorphism optineurin H486R variant previously identified to be associated with glaucoma is selectively impaired in mutant Htt binding. Although optineurin H486R retains the capacity to bind to mGluR1a, optineurin H486R-dependent attenuation of mGluR1a signaling is not enhanced by the expression of mutant Htt. Because G protein-coupled receptor kinase 2 (GRK2) protein expression is relatively low in striatal tissue, we propose that optineurin may substitute for GRK2 in the striatum to mediate mGluR desensitization. Taken together, these studies identify a novel mechanism for mGluR desensitization and an additional biochemical link between altered glutamate receptor signaling and Huntington disease. Huntington disease (HD)4 is an autosomal-dominant neurodegenerative disorder manifested by symptoms of involuntary body movement, loss of cognitive function, and psychiatric disturbance, which inevitably leads to death (1-4). The HD gene mutation consists of an unstable CAG repeat resulting in a polyglutamine expansion in the amino-terminal region of the huntingtin (Htt) protein, a ubiquitously expressed and evolutionary conserved protein (1). It is the polyglutamine expansion of the Htt amino terminus that is proposed to cause progressive widespread neuronal death in the neocortex and the striatum of HD patients. Although the precise function of Htt in cells is not completely understood, analysis of the proteins with which Htt interacts suggests that Htt plays a role in regulating clathrin-coated vesicle-mediated endocytosis, neuronal survival, vesicle transport, morphogenesis, calcium homeostasis, and transcriptional regulation (4).Glutamate-mediated neurotoxicity has been postulated to play an important role in the pathogenesis and excitotoxic neuronal cell loss in HD (5-9). The receptors for glutamate are classified into two types: ionotropic and metabotropic (10). The ionotropic receptors comprise cation-specific ion channels that mediate fast excitatory glutamate responses and are subdivided into AMPA/kain...

Background: CRFR1 regulates the physiological response to stress and is implicated in the manifestation of depression. Results: SAP97 interacts and co-localizes with CRFR1, suppresses CRFR1 endocytosis, and is required for CRFR1-mediated ERK1/2 phosphorylation. Conclusion: SAP97 functionally regulates CRFR1 trafficking and signaling. Significance: This is the first documentation of functional regulation of CRFR1 by a specific PDZ protein.

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BackgroundAgonist stimulation of Group I metabotropic glutamate receptors (mGluRs) initiates their coupling to the heterotrimeric G protein, Gαq/11, resulting in the activation of phospholipase C, the release of Ca2+ from intracellular stores and the subsequent activation of protein kinase C. However, it is now recognized that mGluR5a also functions as a receptor for cellular prion protein (PrPC) and β-amyloid peptide (Aβ42) oligomers to facilitate intracellular signaling via the resulting protein complex. Intracellular mGluR5a signaling is also regulated by its association with a wide variety of intracellular regulation proteins.ResultsIn the present study, we utilized mass spectroscopy to identify calmodulin kinase IIα (CaMKIIα) as a protein that interacts with the second intracellular loop domain of mGluR5. We show that CaMKIIα interacts with both mGluR1a and mGluR5a in an agonist-independent manner and is co-immunoprecipitated with mGluR5a from hippocampal mouse brain. CaMKIIα positively regulates both mGluR1a and mGluR5a endocytosis, but selectively attenuates mGluR5a but not mGluR1a-stimulated ERK1/2 phosphorylation in a kinase activity-dependent manner. We also find that Aβ42 oligomers stimulate the association of CaMKIIα with mGluR5a and activate ERK1/2 in an mGluR5a-dependent manner. However, Aβ42 oligomer-stimulated ERK1/2 phosphorylation is not regulated by mGluR5a/CaMKIIα interactions suggesting that agonist and Aβ42 oligomers stabilize distinct mGluR5a activation states that are differentially regulated by CaMKIIα. The expression of both mGluR5a and PrPC together, but not alone resulted in the agonist-stimulated subcellular distribution of CaMKIIα into cytoplasmic puncta.ConclusionsTaken together these results indicate that CaMKIIα selectively regulates mGluR1a and mGluR5a ERK1/2 signaling. As mGluR5 and CaMKIIα are involved in learning and memory and Aβ and mGluR5 are implicated in Alzheimer’s disease, results of these studies could provide insight into potential pharmacological targets for treatment of Alzheimer’s disease.

The angiotensin II type 1 receptor (AT 1 R) is a G␣ q/11 -coupled G protein-coupled receptor that is widely expressed in multiple tissues, including vascular smooth muscle cells, brain, and kidney. Activation of the AT 1 R in vascular smooth muscle cells leads to alterations in actin-based membrane protrusions such as lamellipodia, filopodia, and membrane blebs that ultimately lead to cell migration, which is important for the regulation of vascular tone. In the present study, we examine the role of small G proteins in mediating AT 1 R-induced alterations in membrane dynamics in human embryonic kidney 293 cells. We find that the activation of the AT 1 R with 100 nM angiotensin II results in the rapid formation of membrane blebs at early time points of agonist stimulation that cease within 40 min of agonist stimulation. AT 1 R-stimulated membrane bleb formation is independent of RalA, RalB, Rac1, cdc42, Arf6, and Ras, but it involves RhoA. Furthermore, membrane blebbing activated by the AT 1 R is attenuated in the presence of the ␤-arrestin aminoterminal domain, Ral GDP dissociation stimulator (RalGDS) ␤-arrestin binding domain, and short interfering RNA (siRNA) depletion of ␤-arrestin2. However, siRNA depletion of RalGDS protein did not affect membrane blebbing in response to AT 1 R activation. The inhibition of the downstream RhoA effectors Rho kinase (ROCK) and myosin light chain kinase (MLCK) effectively attenuated AT 1 R-mediated membrane blebbing. Thus, we show that membrane blebbing in response to AT 1 R signaling is dependent on ␤-arrestin2 and is mediated by a RhoA/ ROCK/MLCK-dependent pathway.

Serotonin (5-HT) interacts with a wide variety of 5-HT receptors (5-HTR) of which 5-HT 2A R plays an important target for antidepressant and atypical antipsychotic drugs. The carboxyl-terminal tail of 5-HT 2A R encodes a motif that mediates interactions with PSD-95/disc large/zona occludens (PDZ) domain-containing proteins. In the present study, we found that 5-HT 2A R interacts with synapse-associated protein 97 (SAP97; also known as DLG1) by coimmunoprecipitation in human embryonic 293 (HEK 293) cells and cortical brain lysates. We found that 5-HT 2A R expression results in the recruitment of SAP97 from the cytosol to the plasma membrane and that this recruitment is dependent on an intact 5-HT 2A R PDZ binding motif. We also show that 5-HT 2A R interacts with SAP97 using bioluminescence energy transfer and that overexpression of SAP97 retards 5-HT 2A R endocytosis, Similarly to what has been observed for the corticotropinreleasing factor receptor 1 (CRFR1), SAP97 expression is essential for 5-HT 2A R-stimulated extracellular-regulated protein kinase 1/2 (ERK1/2) phosphorylation by a PDZ interactionindependent mechanism. Moreover, we find that SAP97 is not responsible for CRFR1-mediated sensitization of 5-HT 2A R signaling. Taken together, our studies show that SAP97 plays a conserved role in regulating 5-HT 2A R endocytosis and ERK1/2 signaling, but plays a novel role in regulating 5-HT 2A R G protein coupling.

The angiotensin II type 1 receptor (AT 1 R) plays an important role in cardiovascular function and as such represents a primary target for therapeutic intervention. The AT 1 R has traditionally been considered to be coupled to the activation of phospholipase C (PLC) ␤ via its association with G␣ q/11 , leading to increases in intracellular inositol phosphate (IP) and release of calcium from intracellular stores. In the present study, we investigated whether the small GTPase RalA contributed to the regulation of AT 1 R endocytosis and signaling. We find that neither RalA nor RalB is required for the endocytosis of the AT 1 R, but that RalA expression is required for AT 1 R-stimulated IP formation but not 5-HT 2A receptor-mediated IP formation.AT 1 R-activated IP formation is lost in the absence of Ral guanine nucleotide dissociation stimulator (RalGDS), and requires the ␤-arrestin-dependent plasma membrane translocation of RalGDS. G␣ q/11 small interfering RNA (siRNA) treatment also significantly attenuates both AT 1 R-and 5-HT 2A receptor-stimulated IP formation after 30 min of agonist stimulation. PLC-␦1 has been reported to be activated by RalA, and we show that AT 1 R-stimulated IP formation is attenuated after PLC-␦1 siRNA treatment. Taken together, our results provide evidence for a G protein-coupled recepto-activated and RalGDS/Ral-mediated mechanism for PLC-␦1 stimulation.