| Recent evidence from this laboratory has indicated that the B cell integral membrane protein CD20 functions in calcium entry following B cell antigen receptor (BCR) stimulation. However, little was known about the specific role of CD20 in calcium entry. Several possibilities included: (1) CD20 is a calcium channel, (2) CD20 associates with a channel thereby regulating calcium entry via an adjacent channel, or (3) CD20 initiates signals which modulate a calcium channel. During the course of my thesis work, I generated results that will aid in understanding the role of CD20. Consistent with its potential role as a component of a store-operated calcium channel, I provided evidence that CD20 exists as a tetramer in association with other surface-labelled proteins. I determined that CD20 associates with cell surface immunoglobulin which is the antigen binding component of the BCR. CD20 is localized to cholesterol-rich signalling domains known as lipid rafts and my evidence indicated that it associates with cell surface immunoglobulin within lipid rafts. However, BCR-CD20 association was resistant to cholesterol depletion and raft disruption, indicating that the receptor-channel complex, while residing in rafts, is maintained independently of the cholesterol-rich raft environment. Interestingly, depletion of membrane cholesterol alters the conformation of CD20, however this effect is regulated independently of CD20 raft association. Since CD20 lipid raft localization is required for BCR-mediated calcium entry, the cholesterol-dependent conformation may reflect the regulation of calcium channel-independent properties of CD20. BCR ligation results in the dissociation of cell surface immunoglobulin from CD20 as well as in the subsequent recruitment of several phosphoproteins and calmodulin binding proteins to the CD20 multimeric complex. Future investigations will likely be aimed at identifying and characterizing these proteins to acquire a better understanding of the function of CD20 in modulating BCR mediated signalling events. |
