| Summary: | Integrins are dynamic cell adhesion molecules expressed on the cell-surface which transmit
signals through the plasma membrane, and play vital physiological roles in multicellular
organisms. The β7 integrins α4β7 and αEβ7 are leukocyte-specific, and evolved to form and
maintain the mucosal immune system of the intestine, which is an important immune barrier.
They participate in antigen presentation, leukocyte migration and homing, and other
leukocyte functions. α4β7 binds the mucosal vascular addressin MAdCAM-1, which is
expressed on specialised high endothelial venules at sites of chronic inflammation. The β7
integrins contribute to the initiation and maintenance of inflammation, in particular chronic
inflammatory disease, playing a key role in the migration of leukocytes into inflamed tissue.
It is important to understand integrin signalling mechanisms in order to be able to devise
therapeutic agents that can inhibit integrin function, and thereby attenuate inflammation. The
host laboratory identified a novel cell adhesion regulatory domain (CARD) within the β7
subunit cytoplasmic domain, which plays a key role in regulating β7-mediated cell adhesion.
The CARD comprises the small pseudosymmetrical motif YDRREY, which forms the basis
of the present study.
This study aimed to identify key signalling pathways involved in regulating the cell adhesion
function of the β7 integrins, to identify intracellular ligands that interact with the β7 CARD,
and to characterize their interaction. The adhesion of the thymic lymphoma TK-1 (α4β7+
α4β1-) to MAdCAM-1 was used to investigate signalling via α4β7. A panel of chemical
inhibitors of cell signalling molecules was employed in TK-1 cell adhesion assays, revealing
that JNK, the src family of kinases, and myosin light chain kinases all potentially participate
in controlling α4β7-mediated cell adhesion.
Cell-permeable synthetic peptide technology was employed to investigate structure-function
relationships of the YDRREY CARD motif. Substitution of the flanking tyrosine residues of
the YDRREY motif with phenylalanines revealed that the tyrosines were critical for the
function of the CARD. However, the state of phosphorylation of the flanking tyrosines was
inconsequential. The core DRRE region was also important for CARD function. Mutagenesis
studies involving substitution of the three tyrosine residues in the β7 subunit cytoplasmic
domain to phenylalanines disrupted α4β7-mediated adhesion of TK-1 cells to MAdCAM-1,
reinforcing the role of tyrosine residues in β7 activation.
The tyrosine kinases src and FAK were shown to bind directly to the YDRREY peptide. FAK
and autophosphorylated src was shown to bind to both phosphorylated and nonphosphorylated
forms of YDRREY, while non-phosphorylated src bound only the nonphosphorylated
form. The cytoskeletal proteins α-actinin, and paxillin formed complexes
with FAK and bound to the YDRREY peptide, whereas filamin disrupted src binding. FAK
and src were both present in immunoprecipitates of α4β7. Immunofluorescence studies
combined with confocal microscopy suggested both kinases co-localise with the β7 integrin
within the trailing edge uropod and at sites of pseudo supra-molecular complex (SMAC)
formation.
Heat shock protein (hsp) 70 was also identified as a potential intracellular ligand that
associates indirectly with the β7 subunit. Thus, a synthetic peptide encompassing the fulllength
β7 cytoplasmic domain bound to recombinant hsp70, but only in the presence of a TK-
1 cell lysate. Heat shock using fever-range temperatures activated α4β7-mediated cell
adhesion. A chemical inhibitor of hsp70 prevented Mn2+-induced adhesion of TK-1 cells to
MAdCAM-1, suggesting that hsp70 plays a key role in regulating signalling by α4β7. The
latter results suggested that other stressors might activate α4β7-mediated cell adhesion, and
in accord serum starvation of cultured cells also induced α4β7-mediated cell adhesion to
MAdCAM-1.
In summary, this study has provided novel insights into signalling pathways that regulate
signalling by β7 integrins. Structure-function relationships of the β7 CARD have been
investigated, and kinases that interact with the CARD have been identified. Activation of β7
integrin signalling by stressors including fever-range temperatures and serum starvation, and
identification of hsp70 as a molecule which associates indirectly with α4β7 are novel
discoveries. The information gained provides a platform of results which provide the basis for
generating agents that might have therapeutic potential for treating inflammatory diseases.
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