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 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.