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. 1999 Aug 17;96(17):9792-6.
doi: 10.1073/pnas.96.17.9792.

Myeloid DAP12-associating lectin (MDL)-1 is a cell surface receptor involved in the activation of myeloid cells

A B Bakker  1 , E Baker, G R Sutherland, J H Phillips, L L Lanier
Affiliations

Myeloid DAP12-associating lectin (MDL)-1 is a cell surface receptor involved in the activation of myeloid cells

A B Bakker et al. Proc Natl Acad Sci U S A. .

Abstract

Crosslinking of immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor complexes on a variety of cells leads to their activation through the sequential triggering of protein tyrosine kinases. Recently, DAP12 has been identified as an ITAM-bearing signaling molecule that is noncovalently associated with activating isoforms of MHC class I receptors on natural killer cells. In addition to natural killer cells, DAP12 is expressed in peripheral blood monocytes, macrophages, and dendritic cells, suggesting association with other receptors present in these cell types. In the present study, we report the molecular cloning of the myeloid DAP12-associating lectin-1 (MDL-1), a DAP12-associating membrane receptor expressed exclusively in monocytes and macrophages. MDL-1 is a type II transmembrane protein belonging to the C type lectin superfamily and contains a charged residue in the transmembrane region that enables it to pair with DAP12. Crosslinking of MDL-1/DAP12 complexes in J774 mouse macrophage cells resulted in calcium mobilization. These findings suggest that signaling via MDL-1/DAP12 complexes may constitute a significant activation pathway in myeloid cells.

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Figures

Figure 1
Figure 1
(A) Rescue of DAP12 cell surface expression on 293T FLAG-DAP12 cells transfected with control plasmid or with pJEF14 vector containing mouse MDL-1 cDNA. (B) Nucleotide and predicted amino acid sequence of mouse MDL-1. The sequence of the cDNA encoding the short form of mouse MDL-1 is presented (GenBank accession no. AF139769). The predicted transmembrane region is underlined, with the charged lysine indicated in bold, and potential N-linked glycosylation sites are depicted with an asterisk. (C) Alignment of the predicted protein sequences of the long form of mouse MDL-1 (GenBank accession no. AA186015) and human MDL-1 (GenBank accession no. AF139768). The additional 25 aa in the stalk region of the long form of mouse MDL-1 are underlined.
Figure 2
Figure 2
MDL-1 gene and expression. (A) Total RNA of mouse cell lines was analyzed for MDL-1 transcripts by using the short form of the mouse MDL-1 cDNA as a probe. EL4, KKF1, and BW 5417 are cell lines of T cell origin. P815 and BaF/3 are mastocytoma and pre-B cell lines, respectively. MC1 and MC12 are mast cell lines, J774 is a macrophage cell line, and RBL-2H3 is a rat basophilic leukemia. (Inset) Hybridization with a β-actin probe is shown. (B) PCR amplification of cDNA prepared by reverse transcription of RNA from healthy donor peripheral blood leukocyte subpopulations or primary cultures thereof or from human cell lines. U937, MM6, and THP-1 are cell lines of myeloid origin; TF-1 and K562 are of erythroid/myeloid and erythroid origins, respectively. JY is a B lymphoblastoid cell line, and Jurkat is a T cell leukemia cell line. NK92 and NKL are NK cell lines. Fibroblasts were derived from human foreskin samples. Colo 205 is a colon carcinoma cell line. (C) MDL-1 Southern blot analysis. Human genomic DNA was digested with the indicated enzymes and probed with the human MDL-1 cDNA.
Figure 3
Figure 3
MDL-1 selectively rescues DAP12 cell surface expression. 293T cells stably expressing FLAG-DAP12 or FLAG-FcɛRIγ were transiently transfected with plasmids encoding human CD16 and human MDL-1, respectively. Cell surface expression of FLAG-DAP12 and FLAG-FcɛRIγ was analyzed by flow cytometry by using the anti-FLAG mAb M2.
Figure 4
Figure 4
MDL-1 associates noncovalently with DAP12 on the cell surface. (A) Mouse J774 macrophage cells expressing MDL-1/CD69 or MDL-1/CD69 in which the transmembrane lysine was replaced by an isoleucine (TM K → I) were analyzed by flow cytometry by using isotype control or anti-CD69 mAb Leu-23. (B) J774 MDL-1/CD69 cells (WT) or J774 MDL-1/CD69 TM K → I cells (K → I) were lysed and were immunoprecipitated with control Ig (cIg) or anti-CD69 mAb. Immunoprecipitates were analyzed by SDS/PAGE under reducing conditions, blotted, and probed with affinity-purified rabbit anti-mouse DAP12 antiserum.
Figure 5
Figure 5
MDL-1 receptor induces intracellular Ca2+ mobilization. Indo-1 AM-loaded MDL-1/CD69-transfected J774 cells were incubated with anti-H2-Dd mAb (used as a negative control) (A) or anti-CD69 mAb (B), followed by a crosslinking Ab. Intracellular calcium levels were measured as described in Materials and Methods.
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