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UBC Theses and Dissertations

Compartmentalization and B cell antigen receptor signaling Jackson, Teresa Lynn

Abstract

This thesis focuses on two aspects of compartmentalization with respect to BCR signaling. In the first section, compartmentalization of the BCR to lipid rafts is considered and in the second section the subsequent compartmentalization of BLNK and PLCy to the BCR is considered. Recent studies have suggested that the BCR translocates into lipid rafts in certain B cell stages. Yet these reports have been ambiguous and the mechanisms regulating such translocation have remained elusive. In this thesis it is demonstrated that the BCR can translocate into lipid rafts following BCR cross-linking in the immature B cell lines, WEHI 231 and CH31 (Jackson et al., 2005). Additionally, it is shown that the Igα/β heterodimer, in the absence of the mlgM subunit, can translocate into lipid rafts in the immature B cell line, WEHI 303.1.5 (Jackson et al., 2005). Previous studies have likewise demonstrated that the mlgM subunit, in the absence of Iga/p, can translocate into lipid rafts (Cheng et al., 2001). Together, these findings may be used to help define a structural feature, common to both subunits, involved in mediating lipid raft association. The PLCy pathway is an integral part of the BCR signaling network. Loss-of-function studies have indicated that the BCR is coupled to PLCy via Syk, BTK and BLNK. In this thesis, a non-lymphoid reconstitution system was used to determine if these components are sufficient to couple the BCR to PLCy. From this it was determined that co-expression of the BCR, Syk and BLNK is sufficient to reconstitute BCR-induced PLCy activation in the system. However, this activation is hypothesized to represent only a partial reconstitution of the pathway as neither BLNK nor PLCy are recruited to the plasma membrane upon BCR cross-linking and as PLCy phosphorylation appears very limited. It was hypothesized that this might be due to the absence of BTK; however, further expression of BTK within the system inhibited rather than enhanced PLCy activation. Subsequent investigations determined that BTK is constitutively activated within this system and as such, may be inappropriately affecting the pathway. Additionally, it was hypothesized that the limited reconstitution may be a consequence of the inability to reconstitute BCRinduced BLNK and PLCy membrane recruitment. Thus, BLNK and PLCy were constitutively targeted to the plasma membrane within the system. From this, it was determined that membrane-targeting of PLCy is sufficient to reconstitute BCR-induced, Syk-dependent PLCy activation. In contrast, membrane-targeting of BLNK is not sufficient to reconstitute BCR-induced PLCy membrane recruitment or to enhance BCR-induced PLCy activation within this system. This suggests that there may be an additional defect in the system that is preventing the formation of a functional BCR/BLNK/PLCy signaling complex. Moreover, these findings suggest that there may be a deficit in our current understanding of the BCR/PLCy pathway. In summary, these findings highlight the importance of compartmentalization in BCR signaling both with respect to compartmentalization of the BCR to lipid rafts and the subsequent compartmentalization of BLNK and PLCy to the BCR.

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