The Relevance of the SIRP Protein Family to Signal Transduction and Cell Adhesion
The SIRPs are a recently discovered family of glycoproteins comprising more than\n30 members belonging to the immunoglobulin superfamily. The two different\nstructural subtypes, termed SIRP \u03b1 and SIRP \u03b2, are distinguished by the presence\nor absence of a cytoplasmic domain, respectively. SIRP \u03b11, the first member of\nthe family to be purified, had been characterised as a negative regulator of signal\ntransduction, and transformation assays had suggested that it also had tumour\nsuppressive effects. Little or nothing is known about the possible function of either\nthe other SIRP \u03b1 homologues or the members of the SIRP \u03b2 subtype.\nThe Ig-like domains possessed in the extracellular domains of all SIRPs suggest\nthey have binding partners outside the cell. Cell adhesion experiments using the\nextracellular domains of SIRP family members showed that SIRP \u03b1 have adhesion\nmolecule properties. This led to the identification of CD47 as one ligand for\nSIRP \u03b1, performed in collaboration with others21and confirmed here. Furthermore,\nthese experiments suggested that SIRP \u03b1 molecules have at least one further\nunknown ligand that is not CD47.\nThe discovery that SIRP \u03b1 was a cell adhesion molecule with a regulatory role in\nsignal transduction was expanded by in vitro kinase experiments and experiments\nwith inhibitors of tyrosine kinases. They showed that SIRP \u03b1 associated with more\nthan one kinase activity, and that cytosolic tyrosine kinases, probably of the srcfamily,\nwere necessary for SIRP \u03b1 to regulate tyrosine phosphorylation of a\nreceptor.\nIn contrast to SIRP \u03b1 molecules, proteins belonging to the SIRP \u03b2 subtype remain\nuncharacterised. Therefore a large part of this work concentrates on the SIRP \u03b2\nsubtype, its associated proteins, localisation and possible function in a cell.\nIn vitro association experiments revealed that SIRP \u03b2 is part of a multiprotein\ncomplex at the cell membrane, where SIRP \u03b21 interacted with DAP12, an adaptor\nprotein with a transmembrane domain. DAP12 linked SIRP \u03b2 to a cytosolic\ntyrosine kinase identified as Syk confirmed by western blot and PCR from cDNA\npreparations of the cell lines used in these experiments. The interaction of Syk with the complex required the tyrosine phosphorylation of DAP12. Coligating SIRP\n\u03b2 molecules at the membrane with a SIRP \u03b2-specific monoclonal antibody\nrecruited Syk to DAP12 where it could be activated by treatment with sodium\npervanadate. In vitro kinase assays detected several unknown phosphorylated\nproteins associated with SIRP \u03b2/DAP12/Syk when Syk was activated that may\nrepresent signalling molecules operating downstream of the complex.\nCotransfection experiments showed that SIRP \u03b1 complexed with kinase activities\nthat enabled it to inhibit both DAP12 tyrosine phosphorylation and Syk kinase\nactivity. This suggested that both complexes at some point operated in close\ncontact, so experiments were carried out to localise SIRP proteins in the cell.\nFractionation experiments discovered that SIRP \u03b1 and possibly SIRP \u03b2 could be\ndetected in fractions that contained GPI microdomains, or caveolae. Similar\ninvestigations with the SIRP \u03b21/DAP12 complex revealed that DAP12 was\ndependent upon SIRP \u03b2 for its direction to the plasma membrane where it was\nactivated by tyrosine kinases. Membrane localisation of SIRP \u03b2 was similarly\nreliant upon DAP12 expression, however, further experiments suggested that\nSIRP \u03b2 may be secreted from the cell in the absence of DAP12.\nTo address the potential role of SIRP \u03b21/DAP12 complex in signal transduction,\ncell lines overexpressing SIRP \u03b2 and DAP12 were analysed. Cell death assays\nsuggested that the SIRP \u03b21/DAP12 complex was a negative regulator of induced\ncell death, and that tyrosine kinases might be involved in this regulation. Cells\noverexpressing SIRP \u03b2 and DAP12 showed an enhanced rate of acid production,\ncorresponding to an enhanced rate of glucose metabolism. These observations\nsuggests that, SIRP \u03b21/DAP12 overexpression may be a factor that contributing to\na transformed phenotype, works in opposition to SIRP \u03b1 molecules.\nThis work views the SIRPs as components of a cluster of different proteins at the\ncell membrane that recruit and use other cytosolic proteins, among them tyrosine\nkinases and phosphatases. It shows that SIRP \u03b1 molecules may collaborate with\nSIRP \u03b2 family members to modulate the signals generated by other receptors in\nsignal transduction. This modulation may influence aberrant cellular processes\nthat lead to disease.