The Stalk Domain of NKp30 Contributes to Ligand Binding and Signaling of a Preassembled NKp30-CD3ζ Complex

Abstract

Edited by Peter CresswellThe natural cytotoxicity receptor (NCR) NKp30 (CD337) is a key player for NK cell immunosurveillance of infections and cancer. The molecular details of ligand recognition and its connection to CD3 signaling remain unsolved. Here, we show that the stalk domain ( 129 KEHPQLGAGTVLLLR 143 ) of NKp30 is very sensitive to sequence alterations, as mutations lead to impaired ligand binding and/or signaling capacity. Surprisingly, the stalk domains of NKp30 and NKp46, another NCR employing CD3 for signaling, were not exchangeable without drastic deficiencies in folding, plasma membrane targeting, and/or ligand-induced receptor signaling. Further mutational studies, N-glycosylation mapping, and plasma membrane targeting studies in the absence and presence of CD3 suggest two interconvertible types of NCR-CD3 assemblies: 1) a signaling incompetent structural NKp30-CD3 complex and 2) a ligandinduced signaling competent NKp30-CD3 complex. Moreover, we propose that ligand binding triggers translocation of Arg-143 from the membrane interface into the membrane to enable alignment with oppositely charged aspartate residues within CD3 and activation of CD3-signaling.

Natural Killer Cell Activation Receptor NKp30 Oligomerization Depends on Its N-Glycosylation

“…Impaired oligomerization would then translate into lower apparent binding affinities, when using surface-based methods such as SPR and ELISA, and into impaired signal transduction in cell-based experiments. The role of the stalk domain in CD3ζ-mediated activation of NK cells was thoroughly characterized by Memmer et al [ 7 ] who showed that mutations in the stalk region close to LBD weaken the K D of B7-H6-Fc whereas BAG-6 binding, again, remained mostly unaffected, although NKp30-Fc IgG fusions and SPR detection were used in these experiments. Subsequent reporter cell-based assays showed that Arg143 (end of the NKp30 stalk region) alignment with the aspartate of CD3ζ is required for signal transduction and that this alignment might be achieved by ligand-induced receptor clustering and/or stalk-dependent conformational changes [ 7 ].…”

“…Such an arrangement would bring the membranes of both cells into very close contact, and such an effect could be further potentiated by NKp30 oligomerization. Local deformation of the NK cell plasma membrane caused by the conformational change of the stalk region induced by ligand binding might trigger signal transduction through the CD3ζ chains associated with the NKp30 transmembrane domain thanks to the interaction of CD3ζ Asp36 residue with NKp30 Arg143 residue occurring at plasma membrane which is required for NKp30 signaling [ 7 ].…”

“…Natural killer protein 30 (NKp30; also known as natural cytotoxicity receptor 3, NCR3; or CD337) is an Ig-like activating receptor of NK cells [ 4 , 6 ]. Similar to other members of CD28 protein family, the extracellular part of NKp30 consists of a single N-terminal Ig-like domain, followed by a distinct 15 amino acids long stalk region proximal to the plasma membrane, which is important for receptor signaling [ 7 ]. Most CD28 family members bind proteins of the B7 family [ 8 ]; however, the first molecules that have been shown to interact with NKp30 are heparin and heparin sulfate, that serve as co ligands and only interact with the glycosylated receptor [ 4 , 9 ].…”

Regulation of the Functions of Natural Cytotoxicity Receptors by Interactions with Diverse Ligands and Alterations in Splice Variant Expression

“…Ligand binding and signaling function by NKp30 is highly dependent upon integrity of the membrane proximal stalk region (35). Crystal structures have revealed that NKp30 and NKp44 can form homodimeric structures with NKp30 dimerizing in a head-to-tail fashion to form an I-type Ig-like fold and two NKp44 V-type Ig-like domains form a saddle-shaped dimer with unique disulfide bridging (36, 37).…”

NKp30 ‐ A prospective target for new cancer immunotherapy strategies

“…One particular work demonstrated that, upon binding of B7‐H6, conformational changes in NKp30 enable the translocation of Arg143 to a position deeper in the phospholipid bilayer. As a result, the positively charged arginine residue aligns with the negatively charged aspartate in CD3ζ or FCεRIγ adaptor proteins, which triggers the NK cell response (Memmer et al, 2016).…”