CHIP (C terminus of Hsc-70 interacting protein) is an E3 ligase that links the protein folding machinery with the ubiquitin-proteasome system and has been implicated in disorders characterized by protein misfolding and aggregation. Here we investigate the role of CHIP in protecting from ataxin-1-induced neurodegeneration. Ataxin-1 is a polyglutamine protein whose expansion causes spinocerebellar ataxia type-1 (SCA1) and triggers the formation of nuclear inclusions (NIs). We find that CHIP and ataxin-1 proteins directly interact and co-localize in NIs both in cell culture and SCA1 postmortem neurons. CHIP promotes ubiquitination of expanded ataxin-1 both in vitro and in cell culture. The Hsp70 chaperone increases CHIP-mediated ubiquitination of ataxin-1 in vitro, and the tetratricopeptide repeat domain, which mediates CHIP interactions with chaperones, is required for ataxin-1 ubitiquination in cell culture. Interestingly, CHIP also interacts with and ubiquitinates unexpanded ataxin-1. Overexpression of CHIP in a Drosophila model of SCA1 decreases the protein steady-state levels of both expanded and unexpanded ataxin-1 and suppresses their toxicity. Finally we investigate the ability of CHIP to protect against toxicity caused by expanded polyglutamine tracts in different protein contexts. We find that CHIP is not effective in suppressing the toxicity caused by a bare 127Q tract with only a short hemaglutinin tag, but it is very efficient in suppressing toxicity caused by a 128Q tract in the context of an N-terminal huntingtin backbone. These data underscore the importance of the protein framework for modulating the effects of polyglutamine-induced neurodegeneration. Polyglutamine (poly-Q)4 diseases are a group of neurodegenerative disorders caused by expansion of glutamine-encoding (CAG) n repeats in genes whose sequence is otherwise unrelated (1, 2). One such protein is ataxin-1, where expansion of its N terminus glutamine repeat triggers spinocerebellar ataxia type 1. SCA1 is an adult-onset disorder characterized by loss of motor coordination and balance, which progresses to affect vital brain functions such as breathing and swallowing. Brain dysfunction is in part due to degeneration of cerebellar Purkinje cells, brainstem neurons, and the spinocerebellar tracts. Strong evidence supports the idea of a gain of function mechanism triggered by the poly-Q expansion in ataxin-1 (1, 3-5).One pathological hallmark of poly-Q disorders is the presence of neuronal aggregates (nuclear or cytoplasmic) that contain the poly-Q-expanded protein. These aggregates are found as nuclear inclusions (NIs) in SCA1 neurons, and in addition to aggregated mutant ataxin-1, they also contain components of the protein quality control machinery, e.g. ubiquitin, proteasome subunits, and chaperones. Such quality control proteins are key players in the toxicity of ataxin-1 and other proteins involved in poly-Q diseases (6 -9).Interestingly, high levels of unexpanded ataxin-1 form NIs and cause degenerative phenotypes similar to, but milder t...

Spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of neurodegenerative disorders sharing atrophy of the cerebellum as a common feature. SCA1 and SCA2 are two ataxias caused by expansion of polyglutamine tracts in Ataxin-1 (ATXN1) and Ataxin-2 (ATXN2), respectively, two proteins that are otherwise unrelated. Here, we use a Drosophila model of SCA1 to unveil molecular mechanisms linking Ataxin-1 with Ataxin-2 during SCA1 pathogenesis. We show that wild-type Drosophila Ataxin-2 (dAtx2) is a major genetic modifier of human expanded Ataxin-1 (Ataxin-1[82Q]) toxicity. Increased dAtx2 levels enhance, and more importantly, decreased dAtx2 levels suppress Ataxin-1[82Q]-induced neurodegeneration, thereby ruling out a pathogenic mechanism by depletion of dAtx2. Although Ataxin-2 is normally cytoplasmic and Ataxin-1 nuclear, we show that both dAtx2 and hAtaxin-2 physically interact with Ataxin-1. Furthermore, we show that expanded Ataxin-1 induces intranuclear accumulation of dAtx2/hAtaxin-2 in both Drosophila and SCA1 postmortem neurons. These observations suggest that nuclear accumulation of Ataxin-2 contributes to expanded Ataxin-1-induced toxicity. We tested this hypothesis engineering dAtx2 transgenes with nuclear localization signal (NLS) and nuclear export signal (NES). We find that NLS-dAtx2, but not NES-dAtx2, mimics the neurodegenerative phenotypes caused by Ataxin-1[82Q], including repression of the proneural factor Senseless. Altogether, these findings reveal a previously unknown functional link between neurodegenerative disorders with common clinical features but different etiology.

Dictyostelium discoideum uses G protein-mediated signal transduction for many vegetative and developmental functions, suggesting the existence of G protein-coupled receptors (GPCRs) other than the four known cyclic adenosine monophosphate (cAMP) receptors (cAR1-4). Sequences of the cAMP receptors were used to identify Dictyostelium genes encoding cAMP receptor-like proteins, CrlA-C. Limited sequence identity between these putative GPCRs and the cAMP receptors suggests the Crl receptors are unlikely to be receptors for cAMP. The crl genes are expressed at various times during growth and the developmental life cycle. Disruption of individual crl genes did not impair chemotactic responses to folic acid or cAMP or alter cAMP-dependent aggregation. However, crlA(-) mutants grew to a higher cell density than did wild-type cells and high-copy-number crlA expression vectors were detrimental to cell viability, suggesting that CrlA is a negative regulator of cell growth. In addition, crlA(-) mutants produce large aggregates with delayed anterior tip formation indicating a role for the CrlA receptor in the development of the anterior prestalk cell region. The scarcity of GFP-expressing crlA(-) mutants in the anterior prestalk cell region of chimeric organisms supports a cell-autonomous role for the CrlA receptor in prestalk cell differentiation.

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cAR1, a G protein-coupled receptor (GPCR) for cAMP, is required for the multicellular development of Dictyostelium. The activation of multiple pathways by cAR1 is transient because of poorly defined adaptation mechanisms. To investigate this, we used a genetic screen for impaired development to isolate four dominant-negative cAR1 mutants, designated DN1-4. The mutant receptors inhibit multiple cAR1-mediated responses known to undergo adaptation. Reduced in vitro adenylyl cyclase activation by GTP␥S suggests that they cause constitutive adaptation of this and perhaps other pathways. In addition, the DN mutants are constitutively phosphorylated, which normally requires cAMP binding and possess cAMP affinities that are ϳ100-fold higher than that of wild-type cAR1. Two independent activating mutations, L100H and I104N, were identified. These residues occupy adjacent positions near the cytoplasmic end of the receptor's third transmembrane helix and correspond to the (E/D)RY motif of numerous mammalian GPCRs, which is believed to regulate their activation. Taken together, these findings suggest that the DN mutants are constitutively activated and block development by turning on natural adaptation mechanisms. INTRODUCTIONG protein-coupled receptors (GPCRs) number in the thousands in humans and are responsible for physiological responses to diverse extracellular signals including light, odorants, hormones, chemoattractants, and neurotransmitters. On activation by their ligands (or photons in the case of rhodopsin), these seven transmembrane domain-containing receptors typically activate heterotrimeric G proteins that, in turn, regulate the activities of a variety of downstream effectors. Ligand binding also triggers various desensitization mechanisms which diminish the cell's responsiveness to subsequent stimulation (Strader et al., 1995;Bourne, 1997;Ferguson and Caron, 1998).These systems are highly conserved and found in all eukaryotes including the social amoeba Dictyostelium discoideum, which uses extracellular cAMP signaling and cAMPspecific GPCRs to orchestrate its multicellular development. When deprived of nutrients, this free-living soil amoeba ceases growth and embarks on a 24-h program in which some 10 5 cells aggregate and differentiate to yield a sporeladen fruiting body. The first of four highly homologous cAMP receptors (cARs) expressed during development, cAR1, is essential for aggregation and subsequent development for three principal reasons. First, by transiently activating an adenylyl cyclase (ACA), cAR1 mediates "cAMP relay," the amplification and propagation of cAMP waves, which arise spontaneously every 6 min at aggregation centers. Second, cAR1 plays a direct role in aggregation as it mediates chemotaxis of cells up the cAMP gradient inherent in each oncoming wave. Lastly, cAR1 is responsible for the induction of various aggregation-stage genes including those encoding cAR1 itself, ACA, and G␣2, the G protein ␣-subunit through which cAR1 signals (Parent and Devreotes, 1996). cAR1 governs these di...