Proc. Natl. Acad. Sci. USA Vol. 93, pp. 685-689, January 1996 Cell Biology Characterization of GMP-17, a granule membrane protein that moves to the plasma membrane of natural killer cells following target cell recognition (cytotoxic lymphocytes/TIA-1/GIG-1/NKG7) QUINTUS G. MEDLEY*t, NANCY KEDERSHAi, STEPHEN O'BRIEN*, QUINSHENG TIAN*§, STUART F. SCHLOSSMAN*§, MICHEL STREULI*t, AND PAUL ANDERSON*§ *Division of Tumor Immunology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115; Departments of tPathology and §Medicine, Harvard Medical School, Boston, MA 02115; and :ImmunoGen, Cambridge, MA 02139 Contributed by Stuart F. Schlossman, October 24, 1995 ABSTRACT Cytotoxic lymphocytes are characterized by NK cells and CTLs (4, 5). Following lymphocyte activation, their inclusion of cytoplasmic granules that fuse with the proteins migrating with apparent molecular masses of 28 kDa plasma membrane following target cell recognition. We pre- (p28-TIA-1) and 40 kDa (p40-TIA-1) are also recognized by the viously identified a cytotoxic granule membrane protein des- 2G9 mAb in immunoblots (4). Molecular cloning of p40-TIA-1 ignated p15-TIA-1 that is immunochemically related to an revealed it to be a member of the RNA-recognition motif (RRM) RNA-recognition motif (RRM)-type RNA-binding protein des- family of RNA-binding proteins (5), and in vitro studies suggested ignated p40-TIA-1. Although it was suggested that p15-TIA-1 that p15-TIA-1 might be derived from p40-TIA-1 by proteolysis might be derived from p40-TIA-1 by proteolysis, N-terminal (5). However, as pulse-chase analysis did not support a precur- amino acid sequencing of p15-TIA-1 immunoaffinity purified sor-product relationship between p40-TIA-1 and p15-TIA-1 (4), from a natural killer (NK) cell line by using monoclonal we purified p15-TIA-1 from the granules of a NK cell line (YT) antibody (mAb) 2G9 revealed that p15-TIA-1 is identical to the and determined its N-terminal amino acid sequence. This se- deduced amino acid sequence of NKG7 and GIG-1, cDNAs quence was found to be identical to the predicted N terminus of isolated from NK cells and granulocyte-colony-stimulating a cDNA-deduced structure previously isolated from NK cells and factor-treated mononuclear cells, respectively. Epitope map- granulocyte-colony-stimulating factor (G-CSF)-treated mononu- ping revealed that mAb 2G9 recognizes the C terminus of clear cells isolated from a patient with chronic myelogenous p15-TIA-1 and p40-TIA-1. The deduced amino acid sequence leukemia [designated NKG7 (6) and GIG-1 (7), respectively]. The of p15-TIA-1/NKG7/GIG-1 predicts that the protein pos- C terminus of p15-TIA-1/NKG7/GIG-1 contains a lysosome sesses four transmembrane domains, and immuno-electron targeting motif that forms part of the 2G9 epitope, revealing the microscopy localizes the endogenous protein to the mem- basis for the immunological similarity between p15-TIA-1 and branes of cytotoxic granules in NK cells. Given its subcellular p40-TIA-1. Because pi5-TIA-1/NKG7/GIG-1 is predicted to localization, we propose to rename this protein GMP-17, for contain four transmembrane regions, is localized to the mem- granule membrane protein of 17 kDa. Immunofluorescence branes of cytotoxic granules, and has a predicted molecular mass microscopy offreshly isolated NK cells confirms this granular of about 17 kDa, we have redesignated this antigen GMP-17, for localization. Target cell-induced NK cell degranulation results granule membrane protein of 17 kDa. Analysis of the subcellular in translocation of GMP-17 from granules to the plasma localization of GMP-17 during activation of NK cells reveals that membrane, suggesting a possible role for GMP-17 in regulat- GMP-17 is relocalized from granules to the plasma membrane, ing the effector function of lymphocytes and neutrophils. suggesting that GMP-17 may contribute to the effector function of cytotoxic lymphocytes. Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) are characterized by their inclusion of cytoplasmic granules that are released in response to target cell recognition. Cyto- MATERIAL AND METHODS lytic lymphocyte granules are morphologically complex or- GMP-17 Purification. All purification procedures were ganelles that can either fuse with the plasma membrane, like done at 4°C or on ice. About 6 x 109 YT cells (kindly provided secretory granules, or fuse with phagosomes, like primary by K. Smith, Cornell University, New York) were sonicated in lysosomes. Although cytotoxic granules are somewhat heter- 40 ml of lysis buffer (50 mM Tris-HCl, pH 7.5/150 mM NaCl/1 ogeneous in structure, they typically contain one or more mM EDTA/1 mM phenylmethanesulfonyl fluoride containing dense cores which are surrounded by multiple small internal aprotinin at 10 ,tg/ml and leupeptin at 50 ,ug/ml) and centri- vesicles (1, 2). The dense cores are the major reservoirs of fuged at 100,000 x g for 30 min. The pellet was resuspended secretory proteins such as perforin, granzymes, and chon- in lysis buffer containing 1% (vol/vol) Nonidet P-40 (NP-40) droitin sulfate proteoglycans (1). In the multivesicular domain, and centrifuged at 3000 x g for 10 min. The supernatant was which resembles endosomally derived multivesicular bodies adjusted to 0.1% SDS and centrifuged at 100,000 x g for 30 found in many cell types, lysosomal membrane proteins such min. The supernatant was precleared for 1 hr with 0.3 ml of as lamp-1, lamp-2, and lgp-120 are abundantly expressed (1). Sepharose CL-4B beads (Pharmacia) and passed over -0.3 ml At the C terminus of these proteins is a conserved motif that of CNBr-activated Sepharose coupled to 2G9 mAb at 5 mg/ml is required for their delivery to lysosome or granule mem- of packed beads. The resin was washed six times in buffer A branes (3). We have characterized an 15-kDa granule mem- (1% NP-40/0.5% sodium brane deoxycholate/0.2% SDS/25 mM protein, designated p15-TIA-1, that is recognized by Tris HCl, pH 7.5/0.5 M NaCl) and the bound protein was 2G9, a monoclonal antibody (mAb) that stains the granules of eluted at 37°C in 2x SDS/PAGE sample buffer containing 0.5 The publication costs of this article were defrayed in part by page charge Abbreviation: CTL, cytotoxic T lymphocyte; NK, natural killer; NP- payment. This article must therefore be hereby marked "advertisement" in 40, Nonidet P-40; mAb, monoclonal antibody; G-CSF, granulocyte- accordance with 18 U.S.C. §1734 solely to indicate this fact. colony-stimulating factor; HA, hemagglutinin. 685 Downloaded by guest on October 1, 2021 686 Cell Biology: Medley et al. Proc. Natl. Acad. Sci. USA 93 (1996) A 1 2 3 4 5 kDa 1 1 1 1 FIG. 1. Comparison of 2G9 mAb-reactive 17- 46 - kDa protein derived from YT cells and GMP-17 expressed in COS-7 cells. (A) YT cells (lanes 1 and 2) and COS-7 cells transfected with plasmid vector pMT.2 (lane 3) or pMT. GMP-17 (lanes 4 and 5) B C were metabolically labeled with [35S]methionine and [35S]cysteine, lysates were prepared, and then pro- - :: 30 .01.1 teins were immunoprecipitated with 2G9 mAb (lanes ,., ';.-A ,i. :,. i:. f:.T 2, 3, and 5) or control IgG mAb (lanes 1 and 4). I Immunoprecipitates were separated in an SDS/14% polyacrylamide gel. Molecular mass markers (kDa) are on the left. (B and C) Metabolically labeled 2G9 mAb-reactive protein from YT cells and recombi- 22 - nant GMP-17 from COS-7 cells were transferred to f._i ... _ nitrocellulose and digested with chymotrypsin. Pep- C tides derived from endogenous p17 (B) and recom- binant p17 (C) were dried and separated by electro- 14 - a 2 phoresis (arrow E) at pH 1.9 and ascending chro- E 4 E J matography (arrow C). M 2-mercaptoethanol. The eluted protein was separated by phytohemagglutinin-activated T-cell library DNA (Clontech). electrophoresis under reducing conditions in an SDS/14% GMP-17 cDNA was inserted into plasmid vectors prior to DNA polyacrylamide gel and then blotted onto a poly(vinylidene sequence determination by the dideoxy chain-termination difluoride) (PVDF) membrane (Applied Biosystems). Follow- method. ing blotting, the membrane was rinsed with water and stained Cell Transfection and Labeling. The expression vectors with amido black, and the membrane portion containing the pMT.2 and pMT.HA containing the GMP-17 cDNA were trans- 17-kDa protein was excised for amino acid sequence analysis. fected into COS-7 cells essentially as described (8). YT cells and N-terminal sequence analysis was performed at the Worcester COS-7 cells were metabolically labeled with [35S]methionine and Microsequencing Facility (Worcester, MA) with an Applied [35S]cysteine, and then solubilized in lysis buffer containing 1% Biosystems model 494 Procise sequencer. NP-40. Protein was immunoprecipitated with 2G9 mAb, the Isolation of GMP-17 cDNA. A GMP-17 cDNA clone was anti-influenza hemagglutinin (HA) mAb 12CA5, or an isotype- isolated by PCR cloning using appropriate primers derived matched control mAb essentially as described (8). The relative from the GIG-1 sequence (ref. 7; accession no. JC2081) and a amount of GMP-17 protein immunoprecipitated was determined A GMP-17 1 . ECRS LG ISC F ....... AVGPTH S T MP20 --. Y M &,F C-X- LL H ....... QYRLSGS F QRY CACNLG 1 MS QT MIIVRVTWFC I LA LAT] AVL S PHMEHHNTTCEEFS I I- TM I--- I GMP-17 46 GH. - .lIMGY- HVT3TGM MP20 46 CLKENCFCQES-Y-EATRF MS CACNLG 57 CTKRIPMDDlKTCGPITLPGEKNCSYFRHFNPGESSEIFEFTTQKEZSa3 GOMP-17 66 wSCFP.... PPGHG VETDAA TSERW MP2O 69 as GGVLAPA RL SR F GI F .