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We examined the role of cysteine (Cys) residues present in chemokine receptor CXCR2 for proper surface expression, dimerization, signaling, and chemotaxis. To address this issue, serine or leucine residues were substituted for Cys, generating nine CXCR2 mutants transiently expressed in HEK cells. Single substitution of Cys residues present in the three extracellular loops (C119L, C196L, C286S) or in the seventh-transmembrane (TM) domain (C308L) abolished CXCL8 agonist binding, while no Cys substitution abolished surface receptor expression. We have previously demonstrated that CXCR2 dimerizes under reducing conditions, due to hydrophobic interactions that involve TM3 regions, and here we show that the dimer/monomer CXCR2 ratio drastically increases when analyzed under non-reducing conditions. We report that none of the Cys-deficient CXCR2 mutants abolishes receptor dimerization, demonstrating that Cys-Cys bonds are not the exclusive determinant of CXCR2 dimerization. Furthermore, both wt- and Cys-mutated CXCR2 dimers are expressed at the cell surface, indicating that receptor dimers are efficiently transferred at the plasma membrane. We also show that every Cys substitution in CXCR2, including those that still bind CXCL8, results in an impairment of receptor activity, analyzed as cell chemotaxis and intracellular signaling, suggesting that some structural requirement is likely fulfilled by Cys presence.

Huntington's disease (HD) is caused by an expanded glutamine tract, which confers a novel aggregation-promoting property on the 350-kDa huntingtin protein. Using specific antibodies, we have probed the structure of the polyglutamine segment in mutant huntingtin complexes formed in cell culture from either truncated or full-length protein. Complexes formed by a mutant amino terminal fragment most frequently entail a change in conformation that eliminates reactivity with the polyglutamine-specific mAb 1F8, coincident with production of insoluble aggregate. By contrast, complexes formed by the full-length mutant protein remain soluble and are invariably 1F8-reactive, indicating a soluble polyglutamine conformation. Therefore, aggregates in HD may form by different biochemical mechanisms that invoke different possibilities for the pathogenic process. If pathogenesis is triggered by a truncated fragment, it probably involves the formation of an insoluble aggregate. However, the observation of soluble complexes in which an HD-specific pathogenic conformation of the glutamine tract remains accessible suggests that pathogenesis could also be triggered at the level of full-length huntingtin by abnormal aggregation with normal or abnormal protein partners., (Copyright 1999 Academic Press.)

A contig covering human chromosome 6p22 that consists of 134 YAC clones aligned based on the presence/absence of 52 DNA markers is presented. This contig overlaps with the 6p23 contig at its telomeric end and with the 6p21.3 contig at its centromeric end. The order of loci within the contig resolves the relative positions of several genetically mapped markers. Among the additional markers used here, there are eight novel PCR assays. The 12 known genes and anonymous ESTs located within the contig establish a first step toward a transcriptional map of this region. The instability of YAC clones observed during this work is also discussed.