Abstract
Here we characterize leukocyte mono-Ig-like receptor 7 (LMIR7)/CLM-3 and compare it with an activating receptor, LMIR4/CLM-5, that is a counterpart of an inhibitory receptor LMIR3/CLM-1. LMIR7 shares high homology with LMIR4 in the amino acid sequences of its Ig-like and transmembrane domains. Flow cytometric analysis demonstrated that LMIR4 was predominantly expressed in neutrophils, whereas LMIR7 was highly expressed in mast cells and monocytes/macrophages. Importantly, LMIR7 engagement induced cytokine production in bone marrow-derived mast cells (BMMCs). Although FcRγ deficiency did not affect surface expression levels of LMIR7, it abolished LMIR7-mediated activation of BMMCs. Consistently we found significant interaction of LMIR7-FcRγ, albeit with lower affinity compared with that of LMIR4-FcRγ. Our results showed that LMIR7 transmits an activating signal through interaction with FcRγ. In addition, like LMIR4, LMIR7 synergizes with TLR4 in signaling. Analysis of several chimera receptors composed of LMIR4 and LMIR7 revealed these findings: 1) the transmembrane of LMIR7 with no charged residues maintained its surface expression at high levels in the absence of FcRγ; 2) the extracellular juxtamembrane region of LMIR7 had a negative effect on its surface expression levels; and 3) the strong interaction of LMIR4 with FcRγ depended on the extracellular juxtamembrane region as well as the transmembrane domain of LMIR4. Thus, LMIR7 shares similarities with LMIR4, although they are differentially regulated in their distribution, expression, and function.
Highlights
A new family of paired immunoreceptors has been recently identified and named leukocyte mono-Ig-like receptor (LMIR)3/CMRF-35-like molecules (CLM)/myeloid-associated
Analysis of several chimera receptors composed of LMIR4 and LMIR7 revealed these findings: 1) the transmembrane of LMIR7 with no charged residues maintained its surface expression at high levels in the absence of FcR␥; 2) the extracellular juxtamembrane region of LMIR7 had a negative effect on its surface expression levels; and 3) the strong interaction of LMIR4 with FcR␥ depended on the extracellular juxtamembrane region as well as the transmembrane domain of LMIR4
Homology research has indicated that LMIR7 is similar to LMIR4 in structure: between them, 85% of the Ig-like domain and 87% of the transmembrane domain are identical in amino acid sequences
Summary
Antibodies and Other Reagents—Rat anti-LMIR7 IgG1 monoclonal Ab (mAb) was generated by ACTGen Inc. Relative expression levels of LMIR7 among samples were measured by real-time RT-PCR as described [3]. A cDNA fragment of LMIR7 lacking the signal sequence was tagged with a FLAG or Myc epitope at the N terminus. PMXs-FLAG- or Myc-LMIR4-IP and pMXs-FcR␥IRES-blasticidin (pMXs-FcR␥-IB) were generated as described [2,3,4]. Biochemistry—BMMCs expressing FLAG-tagged LMIR7 or mock were stimulated by 10 g/ml anti-FLAG mAb or mouse IgG1 mAb as control, 50 ng/ml SCF, or 10 g/ml SPE-7 IgE for the indicated time as described [2,3,4, 28]. Anti-LMIR7 mAb or rat IgG1 mAb as control was biotinylated by sulfo-NHS-LC-biotin (Pierce) according to the manufacturer’s instructions. Statistical Analysis—Data are shown as the mean Ϯ S.D., and statistical significance was determined by Student’s t test with p Ͻ 0.05 taken as statistically significant
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