The long term goals are to understand how the intracellular trafficking and regulated plasma membrane expression of complement receptors in human neutrophils relate to their functions in host defense and immunologic homeostasis. The investigators have previously shown that CR1 and CR3 are upregulated during PMN activation in vivo and that there are serious consequences if this is impaired, as in the deficiency of CR3 in newborns' PMNs and the removal of CR1 from PMN in the lungs of cystic fibrosis patients. They have recently focused on the intracellular storage sites of CR1 in resting PMN and the pathways of internalization of CR1 after cell activation, and they propose to continue these lines of investigation.
In Aim 1 they will immunoisolate the CR1 storage vesicles from resting PMNs using antibodies to the cytoplasmic tail of CR1 they have developed. They will determine which other proteins are stored in these vesicles, and study their mechanisms of fusion with plasma membrane fragments, focusing on the role of low molecular weight G-proteins and annexins. This should provide valuable insights into the mechanisms underlying the sequence of events in neutrophil activation by explaining how the translocation of """"""""readily mobilizable"""""""" compartments like the CR1 storage vesicles differs from the exocytosis of traditional primary and secondary granules.
In Aim 2, they will isolate the MultiVesicular Bodies (MVBs) into which CR1 is internalized after cell activation, demonstrate that they are the sties of CR1 degradation and study how CR1 internalized under different conditions (ligation, crosslinking, etc) is processed and degraded. They will also define the relationships between phosphorylation of CR1, its retention in the MultiVesicular Bodies (MVB) and degradation.
In Aim 3, they will use a Rat Basophil Leukemia (RBL) model, in which transfected CR1 has a trafficking pattern similar to that in PMN, to study how the structure of the tail of CR1 determines the regulation of its membrane expression and trafficking. CR1 mutants with truncated tails or with alterations of the putative phosphorylation site will be compared with wild type CR1 in this model. Finally in Aim 4, they will express in COS cells chimeras of CR1 mutants and FcgammaRIIa to understand why the role of CR1 in phagocytosis is primarily passive, and they will test the hypothesis that both its long filamentous extracellular domain and the lack of signalling motifs in its tail contribute to its inability to activate cells. Taken together, these studies should help us gain a better understanding of the trafficking and function of this important receptor.
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