B- and T-cell development both involve activity of the unfolded protein response pathway.

The unfolded protein response (UPR) signaling pathway regulates the functional capacity of the endoplasmic reticulum for protein folding. Beyond a role for UPR signaling during terminal differentiation of mature B cells to antibody-secreting plasma cells, the status or importance of UPR signaling during hematopoiesis has not been explored, due in part to difficulties in isolating sufficient quantities of cells at developmentally intermediate stages required for biochemical analysis. Following reconstitution of irradiated mice with hematopoietic cells carrying a fluorescent UPR reporter construct, we found that IRE1 nuclease activity for XBP1 splicing is active at early stages of T- and B-lymphocyte differentiation: in bone marrow pro-B cells and in CD4(+)CD8(+) double positive thymic T cells. IRE1 was not active in B cells at later stages. In T cells, IRE activity was not detected in the more mature CD4(+) T-cell population but was active in the CD8(+) cytotoxic T-cell population. Multiple signals are likely to be involved in activating IRE1 during lymphocyte differentiation, including rearrangement of antigen receptor genes. Our results show that reporter-transduced hematopoietic stem cells provide a quick and easy means to identify UPR signaling component activation in physiological settings.

The epidermal growth factor receptor juxtamembrane domain has multiple basolateral plasma membrane localization determinants, including a dominant signal with a polyproline core.

The epidermal growth factor (EGF) receptor is located predominantly in the basolateral membrane of polarized epithelia, where it plays a pivotal role during organogenesis and tissue homeostasis. We have shown previously that a 22-amino acid sequence in the EGF receptor juxtamembrane domain contains autonomous sorting information necessary for basolateral localization using the Madin-Darby canine kidney epithelial cell model. The goal of this study was to determine the molecular basis of EGF receptor basolateral membrane expression using site-directed mutagenesis to modify specific residues in this region. We now show that this sequence has two different, functionally redundant basolateral sorting signals with distinct amino acid requirements: one dependent on residues (658)LL(659) conforming to well-characterized leucine-based sorting signals, and a second containing a polyproline core comprising residues Pro(667) and Pro(670) ((667)PXXP(670)). Our data also suggest that Arg(662) contributes to the function of the proline-based signal. (667)PXXP(670) was the dominant signal when both motifs were present and was more effective than (658)LL(659) at overriding strong apical sorting signals located in the same molecule. Site-directed mutations at Arg(662), Pro(667), and Pro(670) were also associated with increased apical expression of full-length EGF receptors, demonstrating for the first time that the juxtamembrane region is necessary for accurate polarized expression of the native molecule.