The structure, regulation and function of the leukocyte β7 integrins and CG-1

Abstract

This thesis examines the structure, regulation and function of the leukocyte β7 integrins and a novel mouse protein termed CG-1. Members of the integrin family of adhesion molecules are involved in cell-cell and cell-matrix interactions, and play fundamental roles in diverse biological processes. The β7 integrins (LPAM-1, HML-1 and M290) appear to play a special role in mucosal immunity and inflammation. They bind to cell adhesion molecules (CAMs) including MAdCAM-1, VCAM-1 and fibronectin which are expressed on inflamed endothelia and epithelia, and thereby contribute to the processes of leukocyte emigration and retention. An analysis of the β7 gene promoter was undertaken to understand the regulation of expression of the β7 integrins on different leukocyte subtypes. Primer extension and rapid amplification of cDNA ends (RACE) analysis revealed that the start of transcription was confined to nucleotide positions +1 and +4. The 5’ site contains the highly conserved CA motif found at the transcriptional initiation sites of eukaryotic genes. Transient transfection assays revealed that a small 292bp 5’-flanking region of the β7 gene is responsible for cell-specific transcription. This promoter appears to be very compact with an array of potential and often overlapping cis-elements clustered around the transcription start site. The types of cis-elements in the β7 gene promoter are reminiscent of those found in the transcriptional enhancers of the TCR, CD3, integrin α4 subunit, and integrin Leu-CAM genes, in accord with the predominantly leukocyte-restricted distribution of the β7 integrins in normal cells. However, proximal promoter activity was quite weak, and it is likely that distal enhancer elements are required to drive expression of the β7 gene. The presence of an enhancer region located between nucleotide positions -690 and -397 was detected by both deletion analysis and DNase I hypersensitivity assay using the EL4 cell line. Functional studies of the trans-acting factors which bind the cis-acting elements contained within the β7 promoter will aid in unravelling the signals imparted to leukocytes in blood vessels and in gut-associated lymphoid tissues that regulate M290/HML-1 and LPAM-1 expression. CG-1 is a coiled-coil leukocyte protein that is characterized by having extensive heptad repeats. It is immunologically related to, associated with, or identical to CD100, a cell surface antigen which shares a costimulatory function with integrins. CD100 influences the proliferation and differentiation of immune cells. Complementary DNAs encoding the mouse homologue of CG-1 were obtained by screening various mouse cDNA libraries with a human CG-1 cDNA probe. The deduced amino acid (aa) sequence of mouse CG-1 displays 83% identity with the human homologue, and the coiled-coil regions have been stringently conserved. CG-1 transcripts are expressed in a diverse range of cell lines and tissues, and are not restricted to haematopoietic cells. A complex pattern of insertions and/or deletions within CG-1 RNAs was revealed by comparison of the sequences of various mouse cDNA clones isolated from different cells and tissues. The deduced amino acid sequences of mouse and human CG-1 are similar to the sequence of the chicken cardiac morphogenic protein ES/L30, suggesting that CG-1 and ES/130 are potentially related members of a gene family.