The Relevance of the SIRP Protein Family to Signal Transduction and Cell Adhesion

Cant, Charles Alexander (2001): The Relevance of the SIRP Protein Family to Signal Transduction and Cell Adhesion. Dissertation, LMU München: Fakultät für Biologie

Abstract

The SIRPs are a recently discovered family of glycoproteins comprising more than 30 members belonging to the immunoglobulin superfamily. The two different structural subtypes, termed SIRP α and SIRP β, are distinguished by the presence or absence of a cytoplasmic domain, respectively. SIRP α1, the first member of the family to be purified, had been characterised as a negative regulator of signal transduction, and transformation assays had suggested that it also had tumour suppressive effects. Little or nothing is known about the possible function of either the other SIRP α homologues or the members of the SIRP β subtype. The Ig-like domains possessed in the extracellular domains of all SIRPs suggest they have binding partners outside the cell. Cell adhesion experiments using the extracellular domains of SIRP family members showed that SIRP α have adhesion molecule properties. This led to the identification of CD47 as one ligand for SIRP α, performed in collaboration with others21and confirmed here. Furthermore, these experiments suggested that SIRP α molecules have at least one further unknown ligand that is not CD47. The discovery that SIRP α was a cell adhesion molecule with a regulatory role in signal transduction was expanded by in vitro kinase experiments and experiments with inhibitors of tyrosine kinases. They showed that SIRP α associated with more than one kinase activity, and that cytosolic tyrosine kinases, probably of the srcfamily, were necessary for SIRP α to regulate tyrosine phosphorylation of a receptor. In contrast to SIRP α molecules, proteins belonging to the SIRP β subtype remain uncharacterised. Therefore a large part of this work concentrates on the SIRP β subtype, its associated proteins, localisation and possible function in a cell. In vitro association experiments revealed that SIRP β is part of a multiprotein complex at the cell membrane, where SIRP β1 interacted with DAP12, an adaptor protein with a transmembrane domain. DAP12 linked SIRP β to a cytosolic tyrosine kinase identified as Syk confirmed by western blot and PCR from cDNA preparations of the cell lines used in these experiments. The interaction of Syk with the complex required the tyrosine phosphorylation of DAP12. Coligating SIRP β molecules at the membrane with a SIRP β-specific monoclonal antibody recruited Syk to DAP12 where it could be activated by treatment with sodium pervanadate. In vitro kinase assays detected several unknown phosphorylated proteins associated with SIRP β/DAP12/Syk when Syk was activated that may represent signalling molecules operating downstream of the complex. Cotransfection experiments showed that SIRP α complexed with kinase activities that enabled it to inhibit both DAP12 tyrosine phosphorylation and Syk kinase activity. This suggested that both complexes at some point operated in close contact, so experiments were carried out to localise SIRP proteins in the cell. Fractionation experiments discovered that SIRP α and possibly SIRP β could be detected in fractions that contained GPI microdomains, or caveolae. Similar investigations with the SIRP β1/DAP12 complex revealed that DAP12 was dependent upon SIRP β for its direction to the plasma membrane where it was activated by tyrosine kinases. Membrane localisation of SIRP β was similarly reliant upon DAP12 expression, however, further experiments suggested that SIRP β may be secreted from the cell in the absence of DAP12. To address the potential role of SIRP β1/DAP12 complex in signal transduction, cell lines overexpressing SIRP β and DAP12 were analysed. Cell death assays suggested that the SIRP β1/DAP12 complex was a negative regulator of induced cell death, and that tyrosine kinases might be involved in this regulation. Cells overexpressing SIRP β and DAP12 showed an enhanced rate of acid production, corresponding to an enhanced rate of glucose metabolism. These observations suggests that, SIRP β1/DAP12 overexpression may be a factor that contributing to a transformed phenotype, works in opposition to SIRP α molecules. This work views the SIRPs as components of a cluster of different proteins at the cell membrane that recruit and use other cytosolic proteins, among them tyrosine kinases and phosphatases. It shows that SIRP α molecules may collaborate with SIRP β family members to modulate the signals generated by other receptors in signal transduction. This modulation may influence aberrant cellular processes that lead to disease.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 19. Dezember 2001
• 1. Bericht­erstatter:in: David, Charles