Functional Requirements for Signaling through the Stimulatory and Inhibitory Mouse NKR-P1 (CD161) NK Cell Receptors

Abstract

The NK cell receptor protein 1 (NKR-P1) (CD161) molecules represent a family of type II transmembrane C-type lectin-like receptors expressed predominantly by NK cells. Despite sharing a common NK1.1 epitope, the mouse NKR-P1B and NKR-P1C receptors possess opposing functions in NK cell signaling. Engagement of NKR-P1C stimulates cytotoxicity of target cells, Ca2+ flux, phosphatidylinositol turnover, kinase activity, and cytokine production. In contrast, NKR-P1B engagement inhibits NK cell cytotoxicity. Nonetheless, it remains unclear how different signaling outcomes are mediated at the molecular level. Here, we demonstrate that both NKR-P1B and NKR-P1C associate with the tyrosine kinase, p56lck. The interaction is mediated through the di-cysteine CxCP motif in the cytoplasmic domains of NKR-P1B/C. Disrupting this motif leads to abrogation of both stimulatory and inhibitory NKR-P1 signals. In addition, mutation of the consensus ITIM (LxYxxL) in NKR-P1B abolishes both its Src homology 2-containing protein tyrosine phosphatase-1 recruitment and inhibitory function. Strikingly, engagement of NKR-P1C on NK cells obtained from Lck-deficient mice failed to induce NK cytotoxicity. These results reveal a role for Lck in the initiation of NKR-P1 signals, and demonstrate a requirement for the ITIM in NKR-P1-mediated inhibition.

Functional characterization of a conserved pair of NKR‐P1 receptors expressed by NK cells and T lymphocytes in liver and gut

“…NKR-P1F contains a transmembrane arginine that indicates that it may interact with an ITAM-containing adaptor such as FcRγ [32], but previous attempts to demonstrate an activating function for mouse NKR-P1F has been inconclusive [31]. As shown in Figure 2A, we observed a distinct Ca 2+ -response in NK cells in response to NKR-P1F cross-linking, but weaker than NKR-P1A.…”

CD161 (Human NKR-P1A) Signaling in NK Cells Involves the Activation of Acid Sphingomyelinase

“…The molecular mechanisms of signal transduction via human NKR-P1A are unclear. The cytoplasmic tail of human NKR-P1A does not contain the CxCP/S/T lck-binding motif found in CD4, CD8, and rodent NKR-P1 (4,5,14). Although it has been reported that human NKR-P1A can be found in association with src family kinases, including p56 lck (17), others report a failure to reproduce these observations (5).…”

“…Immunoreactive bands were developed using HRP-conjugated secondary Abs (DakoCytomation) and chemiluminescent substrate (ECL Plus; Amersham Biosciences). For assays of enzymatic activity, the beads were equilibrated in aSMase or nSMase buffer, without detergent, and then incubated for 2 h at 37°C in a final volume of 150 ml in the presence of 454 pmol of [N-methyl- 14 C]sphingomyelin (specific activity 55 mCi/mmol; Amersham Biosciences). Radioactive phosphocholine produced from 14 C-labeled sphingomyelin was extracted and quantitated as indicated below.…”

“…Cell lysates were centrifuged for 5 min at 800 ϫ g, and supernatants used for enzymatic activity measurements. Protein content was measured (Coomassie Plus protein assay reagent; Pierce) and equal amount of proteins (50 -80 g) from cellular lysates were added to a final volume of 180 l containing 454 pmol of [N-methyl- 14 C]sphingomyelin and incubated for 2 h at 37°C in aSMase or nSMase buffer. Radioactive phosphocholine produced from 14 C-labeled sphingomyelin was extracted with 1 ml of chloroform to methanol (2/1 v/v) and 400 l of water.…”

“…Different rodent NKR-P1 molecules use different signal transduction pathways to achieve their different functions. The cytoplasmic tail of rodent NKR-P1s has been reported to associate with src family kinases such as p56 lck and various heterotrimeric G proteins (12)(13)(14); whereas the activatory NKR-P1C molecule associates with the ␥-chain of the Fc⑀R (15) and the inhibitory isoform NKR-P1B (expressed by NK cells of SJL/J mice) recruits Src homology protein 1 on cross-linking (7,16). The molecular mechanisms of signal transduction via human NKR-P1A are unclear.…”