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Direct Supervisor: Marcus Thelen, PhD
Location: Laboratory of Signal Transduction
Descriptive title of research activity: Biochemical characterization of chemokine receptors-dependent pathways regulating cell migration
Overall goals: We are investigating receptor proximal signal transduction which mediates cell polarization, actin remodeling and integrin activation. Here we propose to isolate and characterize activated chemokine receptors together with associated proteins to elucidate candidate pathways that regulate actin polymerization.
Rationale and Significance: During embryogenesis cell migration is important for correct cell positioning. In adults leukocyte trafficking is essential for the development of innate and adaptive immune responses. Chemokine receptors also mediate tumor spreading and are crucial for the engraftment of metastases. CXCR4 is known to be widely expressed on human tissues and to mediate such responses. On the other hand, chemokine receptors can be distinguished from each other by their capacity to trigger different pathways. Recent investigations indicate that the activity of CXCR4 could be regulated by not further characterized specific receptor-associated proteins. Thus, elucidation of proximal chemokine receptor-mediated signal transduction may unravel new targets which control cell migration under selective conditions.
Description of work and methodology: Solubilization of heptahelical receptors usually causes their partial inactivation and loss of ligand binding activity. To circumvent this problem we have elaborated a protocol which maintains CXCR4 in its presumed native conformation and might therefore be suitable for the analysis of the 'receptor proteome'. The approach should lead to the identification of proteins that physically interact with and potentially regulate the activity of CXCR4. In preliminary experiments we intend to metabolically label cells with 35S-methionine and determine receptor associated proteins in co-immunoprecipitation assays using the conformational sensitive MoAb 12G5. The experiments will be performed with the T cell line (CEMx174). Our mild solubilization conditions can favor the co-isolation of unspecific contaminants during immunoprecipitation. Therefore protocols will be established to specifically elute receptor complexes from immobilized antibodies and to reduce background signals. A circular peptide is available (T134) which competes for 12G5 binding on CXCR4 and can be used for elution. Alternatively chemical crosslinking can be employed during the separation. It is conceivable that following solubilization depending on their affinity protein complexes ('receptorsomes') remain transiently associated, but are physically separated from irrelevant proteins. Application of cleavable crosslinkers under such conditions will preferentially stabilize preexisting complexes. Such chemically modified protein aggregates can be treated more rigorously to remove contaminants. Immunoprecipitation and separation by non-reducing SDS-PAGE will reveal complexes which can be disintegrated following reduction of the cleavable crosslinker in a second electrophoretic dimension. Isolated proteins will finally be identified by mass spectrometry. We propose to investigate the role of chemokine receptor-dependent phosphorylation of P-Rex1 and to identify the upstream kinase(s). Phosphorylation sites will be mutated by standard molecular biology techniques. Expression of mutated proteins in appropriate cells will show the effect on their subcellular localization and actin polymerization. In vitro assays using purified recombinant proteins will be used to assess the effect of phosphorylation on P-Rex1 activity. We expect that these studies will help to close the gap in knowledge on signal transduction from receptors to actin polymerization.
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