Transmembrane adaptor proteins (TRAPs) are important organisers for the transduction of immunoreceptor-mediated signals. Prr7 is a TRAP that regulates T cell receptor (TCR) signalling and potently induces cell death when overexpressed in human Jurkat T cells. Whether endogenous Prr7 has a similar functional role is currently unknown. To address this issue, we analysed the development and function of the immune system in Prr7 knockout mice. We found that loss of Prr7 partially impairs development of single positive CD4+ T cells in the thymus but has no effect on the development of other T cell subpopulations, B cells, NK cells, or NKT cells. Moreover, Prr7 does not affect the TCR signalling pathway as T cells derived from Prr7 knockout and wild-type animals and stimulated in vitro express the same levels of the activation marker CD69, and retain their ability to proliferate and activate induced cell death programs. Importantly, Prr7 knockout mice retained the capacity to mount a protective immune response when challenged with Listeria monocytogenes infection in vivo. In addition, T cell effector functions (activation, migration, and reactivation) were normal following induction of experimental autoimmune encephalomyelitis (EAE) in Prr7 knockout mice. Collectively, our work shows that loss of Prr7 does not result in a major immune system phenotype and suggests that Prr7 has a dispensable function for TCR signalling.

• Publication type
• Journal Article MeSH

Transmembrane adaptor proteins are membrane-anchored proteins consisting of a short extracellular part, a transmembrane domain, and a cytoplasmic part with various protein-protein interaction motifs but lacking any enzymatic activity. They participate in the regulation of various signaling pathways by recruiting other proteins to the proximity of cellular membranes where the signaling is often initiated and propagated. In this work, we show that LST1/A, an incompletely characterized protein encoded by MHCIII locus, is a palmitoylated transmembrane adaptor protein. It is expressed specifically in leukocytes of the myeloid lineage, where it localizes to the tetraspanin-enriched microdomains. In addition, it binds SHP-1 and SHP-2 phosphatases in a phosphotyrosine-dependent manner, facilitating their recruitment to the plasma membrane. These data suggest a role for LST1/A in negative regulation of signal propagation.

• MeSH
• Cell Membrane metabolism   MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• Major Histocompatibility Complex physiology   MeSH
• Intracellular Signaling Peptides and Proteins MeSH
• Jurkat Cells MeSH
• Humans MeSH
• Membrane Proteins chemistry   genetics   metabolism   MeSH
• Molecular Sequence Data MeSH
• Myeloid Cells cytology   metabolism   MeSH
• Plakins metabolism   MeSH
• Primary Cell Culture MeSH
• Pseudopodia metabolism   MeSH
• Amino Acid Sequence MeSH
• Signal Transduction physiology   MeSH
• Protein Structure, Tertiary physiology   MeSH
• Protein Transport physiology   MeSH
• Protein Tyrosine Phosphatase, Non-Receptor Type 11 metabolism   MeSH
• Protein Tyrosine Phosphatase, Non-Receptor Type 6 metabolism   MeSH
• U937 Cells MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Intracellular Signaling Peptides and Proteins MeSH
• LST1 protein, human MeSH Browser
• Membrane Proteins MeSH
• Plakins MeSH
• PTPN11 protein, human MeSH Browser
• PTPN6 protein, human MeSH Browser
• Protein Tyrosine Phosphatase, Non-Receptor Type 11 MeSH
• Protein Tyrosine Phosphatase, Non-Receptor Type 6 MeSH

BACKGROUND: The importance of membrane compartmentalization into specific membrane microdomains has been shown in many biological processes such as immunoreceptor signaling, membrane trafficking, pathogen infection, and tumor progression. Microdomains like lipid rafts, caveolae and tetraspanin enriched microdomains are relatively resistant to solubilization by some detergents. Large detergent-resistant membrane fragments (DRMs) resulting from such membrane solubilization can be conveniently isolated by density gradient ultracentrifugation or gel filtration. Recently, we described a novel type of raft-like membrane microdomains producing, upon detergent Brij98 solubilization, "heavy DRMs" and containing a number of functionally relevant proteins. Transmembrane adaptor protein LAX is a typical "heavy raft" protein. The present study was designed to identify the molecular determinants targeting LAX-derived constructs to heavy rafts. METHODOLOGY/PRINCIPAL FINDINGS: We prepared several constructs encoding chimeric proteins containing various informative segments of the LAX sequence and evaluated their effects on targeting to heavy rafts. Replacement of the polybasic membrane-proximal part of LAX by CD3ε-derived membrane-proximal part had no effect on LAX solubilization. Similarly, the membrane-proximal part of LAX, when introduced into non-raft protein CD25 did not change CD25 detergent solubility. These results indicated that membrane-proximal part of LAX is not important for LAX targeting to heavy rafts. On the other hand, the replacement of transmembrane part of CD25 by the transmembrane part of LAX resulted in targeting into heavy rafts. We also show that LAX is not S-acylated, thus palmitoylation is not involved in LAX targeting to heavy rafts. Also, covalent dimerization was excluded as a cause of targeting into heavy rafts. CONCLUSIONS/SIGNIFICANCE: We identified the transmembrane domain of LAX as a first motif targeting transmembrane protein constructs to detergent-resistant heavy rafts, a novel type of membrane microdomains containing a number of physiologically important proteins.

• MeSH
• Adaptor Proteins, Vesicular Transport chemistry   genetics   metabolism   MeSH
• Cell Line MeSH
• Protein Interaction Domains and Motifs MeSH
• Humans MeSH
• Membrane Microdomains genetics   metabolism   MeSH
• Protein Multimerization MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adaptor Proteins, Vesicular Transport MeSH
• LAX1 protein, human MeSH Browser