Signal termination processes of GPCRs are well established, unlike processes that regulate the assembly and intracellular trafficking of these signaling complexes. Bimolecular fluorescence complementation was used to study GPCR dimer formation in two projects. Firstly, the importance of Rab GTPases on the cell surface expression and signaling of two chemokine receptors expressed on prostate cancer cells was examined. Rab GTPases necessary for CXCR4 and CCR2 cell surface expression and signaling were different from those necessary for the CXCR4/CCR2 heterodimer. Therefore, this project emphasizes the importance of studying heterodimers as unique entities from their constituent receptors. Secondly, interactions between molecular chaperones and two coreceptors necessary for HIV infection – CCR5, a chemokine GPCR, and the main HIV receptor, CD4, a glycoprotein – were investigated. Further emphasizing the unique characteristics of GPCR dimers, this project found that molecular chaperones interact differently with CCR5 homodimers, when compared to CCR5/CD4 heterodimers.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 132-165.

Thesis (Ph.D. in Cell Biology, Stem Cells, and Development) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 105-116).

The membrane trafficking system mediates delivery of macromolecules and metabolites to discrete intracellular compartments from their site of uptake or synthesis. For many pathogens the trafficking system has a special relevance as it is responsible for maintaining the host-pathogen interface, i.e., the cell surface. Both the surface and the underlying trafficking apparatus are intimately connected with immune evasion in many parasites including those belonging to the highly divergent order Kinetoplastida. Kinetoplastid parasites are etiological agents of several neglected tropical diseases such as African sleeping sickness, Chagas disease, and Leishmaniasis. Newly available sequences of many kinetoplastid genomes were used to reconstruct evolution of trafficking across this lineage, using three central paralogous trafficking families: Rabs, SNAREs and Rab-GAPs, which have defined roles in specific trafficking events. Further, proteomics was used to analyse a representative SNARE complex to explore compositional conservation between kinetoplastids and Opistokhonts. Overall there is little evidence for large scale expansions or contractions of these protein families, excluding a direct association with parasitism or changes to host range, host immunosophistication or transmission mechanisms. The data indicate a stepwise sculpting of the trafficking system where the large repertoire of the basal bodonids is mainly retained by the cruzi group, while extensive lossses characterise other lineages, particularly the African trypanosomes and phytomonads. Kinetoplastids possess several lineage-specific Rabs but all retain a core canonical Rab set; by contrast there is little novelty within the SNARE family even though certain canonical endosomal SNAREs appear to show a considerable degree of sequence divergence. Proteomics suggests that SNARE complex composition is largely conserved. The major changes in Rab and SNARE repertoires are associated with endosomal and late exocytic pathways, which is consistent with the considerable evolution of surface proteomes. Therefore, despite the absence of a transition per se associated with parasitism, adaptation of membrane trafficking is likely under active selection where it meets the host environment.

L'activation constitutive de FGFR3 par mutation ou translocation est l'un des évènements les plus fréquents dans le cancer de la vessie. Une dérégulation de RAB25,une protéine impliquée dans le processus de recyclage des récepteurs de surface, a été montrée dans différents cancers. Des données du transcriptome des cancers de vessie ont montré que RAB25 est surexprimé dans les tumeurs présentant des altérations de FGFR3. L'objectif de cette thèse a été d'étudier l'implication possible de RAB25, des protéines de la même famille, RAB11A et RAB11B, et leur effecteurs RAB11FIP2 et MYO5B dans 1) la tumorigénèse des tumeurs altérées pour FGFR3 et 2) le trafic et la signalisation de FGFR3. Nos résultats montrent que l'extinction de ces protéines par des siARNs induit une diminution significative de la viabilité cellulaire des cellules exprimant des formes constitutivement activées de FGFR3. Les effets de la déplétion de RAB25 et RAB11 sur le recyclage de FGFR3, sur les voies de signalisation de FGFR3 et sur l'expression des gènes cibles de FGFR3 suggèrent que le recyclage de FGFR3 régulé par RAB25 et RAB11 peut prolonger le signal de FGFR3 et peut fournir une plateforme pour la signalisation de FGFR3. Nous avons également comparé la distribution cellulaire des formes sauvage et muté (S249C) de FGFR3 portant une étiquette GFP dans des cellules HeLa. Les deux formes de FGFR3 se trouvent dans plusieurs compartiments intracellulaires mais FGFR3 muté se localise préférentiellement dans le compartiment de recyclage. Ce projet nous a permis de mieux caractériser la trafic de FGFR3 dans le cancer de la vessie et son lien avec la signalisation et l'activité de FGFR3. / Activation of FGFR3 by point mutation, translocation and overexpression is one of the most frequent events in bladder cancer. The dysfunction of RAB25, a GTPase involved in endocytic recycling of transmembrane receptor, has been shown in many cancers. Gene expression data in bladder cancer indicates that RAB25 expression is significant higher in tumors carrying altered FGFR3. The thesis project aimed to investigate the potential role of RAB25, proteins from the same family (RAB11A and RAB11B) and their effectors RAB11FIP2 and MYO5B in 1) the tumorigenesis of tumors carrying altered FGFR3 and 2) the trafficking and the signaling of FGFR3. Our results demonstrate that depletion of these proteins by siRNA significantly reduces cell viability in cells expressing constitutively activated forms of FGFR3. The effects of RAB25 and RAB11 silencing on FGFR3 trafficking and signaling and the expression of FGFR3 target genes suggest that the RAB11- and RAB25-mediated recycling can sustain the signaling by protecting altered FGFR3 from the degradation pathway, and can provide a platform for FGFR3 signaling We also compared the subcellular distribution of wild type and mutant (S249C) forms of FGFR3. These two forms localize to different compartments including early endosomes, late endosomes and recycling compartments. The S249C FGFR3 mutant preferentially localizes to the endocytic recycling compartment. Our findings shed light to the molecular mechanisms underlying the relationships between the trafficking and signaling of FGFR3 in the context of bladder cancer.

Crosspresentation allows antigen presenting cells to present peptides from exogenously derived antigens onto MHC Class I for presentation to CD8+ T cells. Though this pathway shares key players with the Classical Class I and Class II pathways, several questions remain. A genomewide siRNA screen was performed to look for genes that selectively affected the crosspresentation or the Class II pathways. Among the genes identified in the screen was the Rab GTPase Rab39a. Rab39a was required for efficient crosspresentation but was dispensable for the presentation of endogenously expressed antigen. Both TAP-dependent and independent antigen required Rab39a for efficient presentation. Rab39a localized to late endosomes and phagosomes, though interestingly it was not required for the Class II pathway. Analysis of phagosomes from Rab39a KO or rescued cells has shown that in the presence of Rab39a, phagosomes were enriched for the open form of MHC Class I as well as TAP1, a member of the peptide loading complex. The enriched open form of MHC-I was peptide receptive, suggesting that it could contribute to crosspresentation. Phagosomes from Rab39a positive cells had reduced degradative capability and had increased levels of Sec22b, a SNARE protein reported to deliver ER-golgi sourced cargo to phagosomes. Furthermore, inhibition of ER-golgi transport via brefeldin A abolished the phenotype conferred by Rab39a. Thus, we hypothesize that Rab39a mediates the delivery of ER-golgi derived cargo to the antigen containing phagosome. This delivery allows peptide receptive MHC-I, as well as the peptide loading complex to reach the antigen, thereby facilitating crosspresentation.

<p dir="ltr"><i>Legionella pneumophila</i> strains harboring wild-type <i>rpsL</i> such as Lp02<i>rpsL</i>_WT cannot replicate in mouse bone marrow-derived macrophages (BMDMs) due to induction of extensive lysosome damage and apoptosis. The mechanism of this unique infection-induced cell death remains unknown. Using a genome-wide CRISPR/Cas9 screening, we identified <i>Hmg20a </i>and <i>Nol9</i> as host factors important for restricting strain Lp02<i>rpsL</i>_WT in BMDMs. Depletion of <i>Hmg20a</i> protects macrophages from infection-induced lysosomal damage and apoptosis, allowing productive bacterial replication. The restriction imposed by <i>Hmg20a</i> was mediated by repressing the expression of several endo-lysosomal proteins, including the small GTPase Rab7. We found that SUMOylated Rab7 is recruited to the bacterial phagosome via SulF, a Dot/Icm effector that harbors a SUMO-interacting motif (SIM). Moreover, overexpression of Rab7 rescues intracellular growth of strain Lp02<i>rpsL</i>_WT in BMDMs. Our results establish that <i>L. pneumophila</i> exploits the lysosomal network for the biogenesis of its phagosome in BMDMs.</p>

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