The intracellular assembly of immunoglobulins (Ig) in B lineage cells is essential for normal antibody responses and is stringently regulated at multiple levels by endoplasmic reticulum (ER) chaperones. FCRLA is an unusual member of the larger Fc receptor (FCR) and FCR-like (FCRL) gene family and its expression in hemopoietic cells is restricted to the B lineage. In marked contrast to FCRL1-6 and the "classical" Ig-binding FcR, which are transmembrane glycoproteins with extracellular immunoglobulin (Ig) domains, FCRLA is an intracellular protein. It lacks N-linked glycosylation sites and a transmembrane region, but contains a signal sequence to target it to the ER. FCRLA is a resident ER protein that associates with immunoglobulin in this organelle. Unique among Fc receptors, FCRLA can associate with multiple isotypes of Ig, IgM, IgG and IgA. The distinctive features of this protein lead to our central hypothesis: FCRLA is important in the initial metabolism of Ig in B cells and thus in normal immune system function. The significance of the proposal begins with the observation that FCRLA associates with Ig in the ER, which suggests a novel role for this protein in Ig assembly or degradation. Defects in human FCRLA expression may thus lead to autoimmunity or immunodeficiency diseases. The goal of our studies is to define the composition and functions of the Ig- FCRLA complexes. These functions could include aiding Ig folding, facilitating Ig assembly, regulating Ig transport, and/or contributing to the degradation f incompletely processed Ig molecules. FCRLA may thus play an essential role in the intracellular quality control system that ensures the correct production of antibodies. The biochemical and intracellular features of FCRL have so far only been defined in humans and the Fcrla knockout mouse has no perceptible phenotype, therefore the following studies will be done using human B cell lines: 1) Clarify the mechanism for the observed preferential association of FCRLA with the secretory isoform of IgM. 2) Determine the nature of the FCRLA-Ig complexes, including the degree of assembly of the associated Ig, identify any other Ig and/or FCRLA-associated proteins and define changes in the components of the complex that are dependent of the Ig isotype bound and/or the stage of B cell differentiation. 3) Define where along the secretory pathway FCRLA is released from Ig or if the FCRLA bound Ig is destined for destruction. 4) Pinpoint the regions of molecular interaction by mutagenesis of both FCRLA and Ig genes. 5) Ascertain FCRLA function by analysis of phenotypic changes induced by overexpression and shRNA depletion of FCRLA. The analysis here will include Ig assembly, pentamerization and J chain addition in the case of IgM, secretion, cell surface expression, and turnover. Changes in cellular growth rate, cell cycle distribution, and apoptosis will also be determined. These studies are innovative because of the novel features of FCRLA. No other FcR or Fc-like receptor has been shown to associate with IgM, IgG and IgA isotypes. Moreover, FCRLA is the only known Fc-like receptor that is an ER resident Ig-binding protein.
The proposed research is relevant to public health because the regulated assembly and transport of antibodies is essential for protective immune responses to most pathogens. The molecule we have identified and are studying, FCRLA, is positioned in a cellular compartment where it interacts with nascent antibodies and is expected to play an important role in antibody quality control. Therefore these studies are relevant to the NIH mission to foster pursuit of fundamental knowledge and reduce the burdens of human illness.
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