Pathological degeneration of neurons in Huntington's disease and associated neurodegenerative disorders is directly correlated with the expansion of CAG repeats encoding polyglutamines of extended length. The physical properties of extended polyglutamines and the intracellular consequences of expression of polyglutamine expansion have been the object of intensive investigation. We have extended the range of lengths of polyglutamine produced by recombinant DNA methodology by constructing a library of CAG͞CAA repeats coding for a range of 25-300 glutamine residues. We have investigated the subcellular localization, interaction with other polyglutamine-containing polypeptides, and the physical properties of aggregated forms of polyglutamine in the cell. Extended polyQ aggregated in the cytoplasm and was only transported to the nucleus when a strong nuclear localization signal was present. Polyglutamine below pathological lengths could be captured in aggregates and transported to ectopic cell locations. The CREB-binding protein (CBP), containing a homopolymeric stretch of 19 glutamines, was likewise found to coaggregate in a polyglutamine-dependent manner, suggesting that pathology in polyglutamine disease may result from cellular depletion of normal proteins containing polyglutamine. We have observed a striking detergent resistance in aggregates produced from polyglutamine of pathological length. This observation has led to the development of a f luorescence-based assay exploiting the detergent resistance of polyglutamine aggregates that should facilitate high-throughput screening for agents that suppress polyglutamine aggregation in cells.

The transmembrane proto-oncogene receptor tyrosine kinase (RTK) ROS is an orphan receptor that is aberrantly expressed in neoplasms of the central nervous system. Here, we report the fusion of its carboxy-terminal kinase domain to the amino-terminal portion of a protein called FIG (Fused in Glioblastoma) in a human glioblastoma multiforme (GBM). By characterizing both FIG and ROS genes in normal and in U118MG GBM cells, we determined that an intra-chromosomal homozygous deletion of 240 kilobases on 6q21 is responsible for the formation of the FIG-ROS locus. The FIG-ROS transcript is encoded by 7 FIG exons and 9 ROS-derived exons. We also demonstrate that the FIG-ROS locus encodes for an in-frame fusion protein with a constitutively active kinase activity, suggesting that FIG-ROS may act as an oncogene. This is the first example of a fusion RTK protein that results from an intra-chromosomal deletion, and it represents the first fusion RTK protein isolated from a human astrocytoma.

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