OSBP-related protein 3 (ORP3) coupling with VAMP-associated protein A regulates R-Ras activity.

ORP3 is an R-Ras interacting oxysterol-binding protein homolog that regulates cell adhesion and is overexpressed in several cancers. We investigated here a novel function of ORP3 dependent on its targeting to both the endoplasmic reticulum (ER) and the plasma membrane (PM). Using biochemical and cell imaging techniques we demonstrate the mechanistic requirements for the subcellular targeting and function of ORP3 in control of R-Ras activity. We show that hyperphosphorylated ORP3 (ORP3-P) selectively interacts with the ER membrane protein VAPA, and ORP3-VAPA complexes are targeted to PM sites via the ORP3 pleckstrin homology (PH) domain. A novel FFAT (two phenylalanines in an acidic tract)-like motif was identified in ORP3; only disruption of both the FFAT-like and canonical FFAT motif abolished the phorbol-12-myristate-13-acetate (PMA) stimulated interaction of ORP3-P with VAPA. Co-expression of ORP3 and VAPA induced R-Ras activation, dependent on the interactions of ORP3 with VAPA and the PM. Consistently, downstream AktS473 phosphorylation and ß1-integrin activity were enhanced by ORP3-VAPA. To conclude, phosphorylation of ORP3 controls its association with VAPA. Furthermore, we present evidence that ORP3-VAPA complexes stimulate R-Ras signaling.

Attenuated dopaminergic neurodegeneration and motor dysfunction in hemiparkinsonian mice lacking the α5 nicotinic acetylcholine receptor subunit.

Preclinical studies suggest the involvement of various subtypes of nicotinic acetylcholine receptors in the pathophysiology of Parkinson's disease, a neurodegenerative disorder characterized by the death of dopaminergic neurons in the substantia nigra pars compacta (SNC). We studied for the first time the effects of α5 nicotinic receptor subunit gene deletion on motor behavior and neurodegeneration in mouse models of Parkinson's disease and levodopa-induced dyskinesia. Unilateral dopaminergic lesions were induced in wild-type and α5-KO mice by 6-hydroxydopamine injections into the striatum or the medial forebrain bundle. Subsequently, rotational behavior induced by dopaminergic drugs was measured. A subset of animals received chronic treatments with levodopa and nicotine to assess levodopa-induced dyskinesia and antidyskinetic effects by nicotine. SNC lesion extent was assessed with tyrosine hydroxylase immunohistochemistry and stereological cell counting. Effects of α5 gene deletion on the dopaminergic system were investigated by measuring ex vivo striatal dopamine transporter function and protein expression, dopamine and metabolite tissue concentrations and dopamine receptor mRNA expression. Hemiparkinsonian α5-KO mice exhibited attenuated rotational behavior after amphetamine injection and attenuated levodopa-induced dyskinesia. In the intrastriatal lesion model, dopaminergic cell loss in the medial cluster of the SNC was less severe in α5-KO mice. Decreased striatal dopamine uptake in α5-KO animals suggested reduced dopamine transporter function as a mechanism of attenuated neurotoxicity. Nicotine reduced dyskinesia severity in wild-type but not α5-KO mice. The attenuated dopaminergic neurodegeneration and motor dysfunction observed in hemiparkinsonian α5-KO mice suggests potential for α5 subunit-containing nicotinic receptors as a novel target in the treatment of Parkinson's disease.