NKp44 surface expression in COS-7 cell transfectants. (A) Cell surface expression of NKp44 protein in COS-7 cells requires cotransfection with DAP12 cDNA. COS-7 cells were cotransfected with NKp44 ORF and DAP12 cDNAs (upper panels) and stained with three different anti-NKp44 mAbs: Z231, AZ140, and KS38. Lower panels represent COS-7 cells transfected with DAP12 or NKp44 ORF cDNAs alone and stained with the prototype Z231 mAb. Cells were then stained with phycoerythrin–conjugated goat anti–mouse IgG1 or IgM and analyzed by flow cytometry. White profiles represent cells incubated with the second reagent only. Although not shown, the anti-NKp44 mAbs AZ140 and KS38 failed to stain COS-7 cells transfected with NKp44 ORF or DAP12 cDNAs alone. In each panel, the percent of positive cells is indicated; numbers in parentheses indicate the mean fluorescence intensity. (B) Expression of NKp44 and DAP12 mRNA in COS-7 cell transfectants. NKp44 and DAP12 mRNA expression was analyzed by RT-PCR and dot blot using poly A⁺ RNA (see Materials and Methods). On the left, RT-PCR was performed using primers specific for NKp44, DAP12, and β-actin. (Lane 1) Untransfected COS-7 cells; (lane 2) NKp44 transfected COS-7 cells; (lane 3) NKp44/DAP12 cotransfected COS-7 cells; (lane 4) nonretrotranscribed poly A⁺ RNA; (lane 5) NK cell clone LOS64; (lane 6) negative control (PCR reaction mix without cDNA); (lane 7) HinfI digested φX 174 molecular weight markers. On the right, a dot blot prepared using poly A⁺ RNA was hybridized with NKp44, DAP12, and β-actin probes. (lane a) NK cell clone C-11; (lane b) untransfected COS-7 cells; (lane c) mock transfected COS-7 cells; (lane d) NKp44 transfected COS-7 cells; (lane e) NKp44/DAP12 cotransfected COS-7 cells. (C) Biochemical analysis of NKp44 glycoproteins. A polyclonal NK cell population (lanes a and b), and COS-7 cells, untransfected (lane c) or cotransfected with NKp44 and DAP12 cDNAs (lanes d and e), were surface labeled with ¹²⁵I and immunoprecipitated with BAB281 (anti-NKp46) (lanes a and d) or Z231 (anti-NKp44) (lanes b, c, and e) mAbs. Samples were analyzed in a 11% SDS-PAGE under reducing conditions. Molecular mass markers (kD) are indicated on the right.

Association of NKp44 with KARAP/DAP12 ITAM-bearing molecules. (A) 1% digitonin cell lysates derived from a polyclonal NK cell population were immunoprecipitated with either BAB281 (anti-NKp46) or Z231 (anti-NKp44) mAbs. Samples were analyzed in a 15% SDS-PAGE under reducing conditions, transferred to PVDF membranes, and probed with the anti-FcεRIγ antiserum. (B) 1% digitonin cell lysates derived from a polyclonal NK cell population, untreated or treated with sodium pervanadate, were immunoprecipitated with mAbs to the indicated molecules. Samples were analyzed in a 15% SDS-PAGE under reducing conditions, transferred to PVDF membranes, and probed with either the antiphosphotyrosine mAb (anti-PTyr) or the anti-DAP12 antiserum. (C) A polyclonal NK cell population was treated or not treated with KS38 (anti-NKp44) mAb or sodium pervanadate. Cell lysates were immunoprecipitated with Z231 (anti-NKp44) mAb. Samples were analyzed in a 15% SDS-PAGE under reducing conditions, transferred to PVDF membranes, and probed with the antiphosphotyrosine mAb (anti-PTyr). Ig(L), Ig light chains; P-ζ, Tyr-phosphorylated CD3ζ; P-DAP12, Tyr-phosphorylated KARAP/DAP12, and DAP12, unphosphorylated form of KARAP/DAP12 are indicated by arrows. Molecular mass markers (kD) are indicated on the right.

Predicted amino acid sequence and hydrophobicity plot of the NKp44 receptor. (A) Amino acids included in the signal peptide are indicated in small letters. The disulphide bond of the Ig-like V domain is indicated. The potential O-glycosylation sites are marked by single asterisks, and the potential N-glycosylation site by double asterisks. The predicted transmembrane portion is underlined and the charged residue is marked by an arrow. The ITIM sequence is boxed. These sequence data are available from EMBL/GenBank/DDBJ under accession number AJ225109. (B) Kyte-Doolittle hydrophobicity plot of the NKp44 protein.

NKp44 transcript expression in polyclonal and clonal NK cells. Total RNA (5 μg/lane) isolated from the B-EBV cell line LCL721.221, the leukemic T cell line Jurkat, cultured polyclonal NK cell populations (CD, EC, and AM), two NK cell clones (82G16, 82G23), and a CD3⁻ NK cell expansion (LDGLRP) were hybridized with the NKp44 ORF probe. The positions of 28S and 18S ribosomal subunits are indicated. Arrows indicate the two NKp44-specific transcripts. The two bottom insets represent the control hybridization with a β-actin cDNA probe.

Southern blot analysis of NKp44 gene. Genomic DNA (10 μg/lane) extracted from human, monkey, and mouse cells was digested with the indicated restriction enzymes and hybridized with the NKp44 ORF probe. Molecular weight markers (23,130; 9,416; 6,557; 4,361; 2,322; 2,027; 1,353; and 1,078 bp [top to bottom]) are shown on the left.