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Functional requirements for inhibitory signal transmission by the immunomodulatory receptor CD300a.

ABSTRACT: Activation signals can be negatively regulated by cell surface receptors bearing immunoreceptor tyrosine-based inhibitory motifs (ITIMs). CD300a, an ITIM bearing type I transmembrane protein, is expressed on many hematopoietic cells, including subsets of lymphocytes.We have taken two approaches to further define the mechanism by which CD300a acts as an inhibitor of immune cell receptor signaling. First, we have expressed in Jurkat T cells a chimeric receptor consisting of the extracellular domains of killer-cell immunoglobulin-like receptor (KIR)2DL2 fused to the transmembrane and cytoplasmic segments of CD300a (KIR-CD300a) to explore surrogate ligand-stimulated inhibition of superantigen stimulated T cell receptor (TCR) mediated cell signaling. We found that intact CD300a ITIMs were essential for inhibition and that the tyrosine phosphorylation of these ITIMs required the src tyrosine kinase Lck. Tyrosine phosphorylation of the CD300a ITIMs created docking sites for both src homology 2 domain containing protein tyrosine phosphatase (SHP)-1 and SHP-2. Suppression of SHP-1 and SHP-2 expression in KIR-CD300a Jurkat T cells with siRNA and the use of DT40 chicken B cell lines expressing CD300a and deficient in several phosphatases revealed that SHP-1, but not SHP-2 or the src homology 2 domain containing inositol 5' phosphatase SHIP, was utilized by CD300a for its inhibitory activity.These studies provide new insights into the function of CD300a in tuning T and B cell responses.

SUBMITTER: DeBell KE

PROVIDER: S-EPMC3418551 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Functional requirements for inhibitory signal transmission by the immunomodulatory receptor CD300a.

DeBell Karen E KE Simhadri Venkateswara R VR Mariano John L JL Borrego Francisco F

BMC immunology 20120426

<h4>Background</h4>Activation signals can be negatively regulated by cell surface receptors bearing immunoreceptor tyrosine-based inhibitory motifs (ITIMs). CD300a, an ITIM bearing type I transmembrane protein, is expressed on many hematopoietic cells, including subsets of lymphocytes.<h4>Results</h4>We have taken two approaches to further define the mechanism by which CD300a acts as an inhibitor of immune cell receptor signaling. First, we have expressed in Jurkat T cells a chimeric receptor co ...[more]

PMID: 22537350

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Project description:P2X2 receptor channel, a homotrimer activated by the binding of extracellular adenosine triphosphate (ATP) to three intersubunit ATP-binding sites (each located ?50 Å from the ion permeation pore), also shows voltage-dependent activation upon hyperpolarization. Here, we used tandem trimeric constructs (TTCs) harboring critical mutations at the ATP-binding, linker, and pore regions to investigate how the ATP activation signal is transmitted within the trimer and how signals generated by ATP and hyperpolarization converge. Analysis of voltage- and [ATP]-dependent gating in these TTCs showed that: (a) Voltage- and [ATP]-dependent gating of P2X2 requires binding of at least two ATP molecules. (b) D315A mutation in the ?-14 strand of the linker region connecting the ATP-binding domains to the pore-forming helices induces two different gating modes; this requires the presence of the D315A mutation in at least two subunits. (c) The T339S mutation in the pore domains of all three subunits abolishes the voltage dependence of P2X2 gating in saturating [ATP], making P2X2 equally active at all membrane potentials. Increasing the number of T339S mutations in the TTC results in gradual changes in the voltage dependence of gating from that of the wild-type channel, suggesting equal and independent contributions of the subunits at the pore level. (d) Voltage- and [ATP]-dependent gating in TTCs differs depending on the location of one D315A relative to one K308A that blocks the ATP binding and downstream signal transmission. (e) Voltage- and [ATP]-dependent gating does not depend on where one T339S is located relative to K308A (or D315A). Our results suggest that each intersubunit ATP-binding signal is directly transmitted on the same subunit to the level of D315 via the domain that contributes K308 to the ?-14 strand. The signal subsequently spreads equally to all three subunits at the level of the pore, resulting in symmetric and independent contributions of the three subunits to pore opening.

Dataset Information

Functional requirements for inhibitory signal transmission by the immunomodulatory receptor CD300a.

Publications

Functional requirements for inhibitory signal transmission by the immunomodulatory receptor CD300a.

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