is a common KIR3DL1
allele. It is expressed at a frequency of ~ 20% in the Caucasian population and has been shown to provide a selective advantage against AIDS progression (26
appears to be maintained by natural selection, yet its mechanism of action has been unclear, as the translated protein is predominantly retained inside the cell (27
). Here, we report that KIR3DL1*004 is largely misfolded and sequestered in the ER where it binds the molecular chaperone protein calreticulin, suggesting this is a key chaperone involved in KIR3DL1 folding and expression. Intracellular KIR3DL1*004 does not affect HLA-Bw4 ligand expression, nor does it exert an intracellular function by inducing a cellular stress response that might alter NK cell activity. Furthermore, we observed only minimal intracellular interactions between the HLA-Bw4 ligand and the KIR3DL1*002 allotype, which is highly expressed on the cell surface. Together this characterization of intracellular membrane traffic and molecular interactions for KIR3DL1 molecules does not support the hypothesized intracellular function for the KIR3DL1*004 allotype. Rather, the small amount of correctly folded KIR3DL1*004 that makes it to the cell surface is a functionally competent receptor that can transduce an inhibitory signal for NK cell activation. We therefore suggest that the selective advantage for this allele and its ligand is conferred by its capacity to function at the cell surface.
There are two ways in which expression of a low amount of KIR3DL1*004 might influence the outcome of an immune response and it is probable that both of these mechanisms play a role in modulating NK cell function. First, in the effector phase of NK cell activity, a low amount of inhibitory receptor could make the NK cell more readily activated, as observed for KIR3DL1*005 (22
). Second, cell surface KIR3DL1*004 and its putative interaction with HLA-Bw4 could also exert an effect in the process of education during NK cell development (5
). Although the mechanism of NK cell education is incompletely understood, evidence suggests two features. First, the frequency of KIR3DL1/S1 NK cells appears to be positively influenced by ligand expression (64
). Second, it seems that potent receptor activity for education is not simply correlated with abundance on the cell surface. Notably, HLA-C molecules, which are expressed at considerably lower levels than HLA-A or HLA-B (65
), are potent educating ligands upon engagement of the inhibitory KIR2DL receptors (5
). Such observations are consistent with NK cell education being the result of a high avidity interaction that induces a particular signaling pathway favorable for future activation, rather than education relying on a particular signaling threshold determined by abundance of receptor-ligand interactions. The low amount of KIR3DL1*004 could thereby have an effect on NK cell education, depending on its avidity for ligand. Thus, the folding polymorphism that causes retention of the majority of KIR3DL1*004 and cell surface expression of a small amount, might be favorable at the NK cell education stage and reduce the inhibitory signal at the effector stage.
It is also conceivable that low level engagement of KIR3DL1*004 during maturation may influence later NK cell activity by a more subtle mechanism. For example, a heterozygous cell expressing KIR3DL1*004, as well as a more abundant or very avid KIR3DL1 allotype, might receive signals equivalent to a cell homozygous for the co-expressed allele. Selection for KIR3DL1*004 and its ligand would therefore allow the co-expressed KIR3DL1 allotype to dominate during selection. This effect would also be true for other KIR loci encoding variants with reduced or null surface expression. Ultimately, this should be detectable by evidence for selection of heterozygosity for alleles with impaired expression at these loci.
Characterization of membrane traffic pathways is highly relevant to NK cell function because secretory or endocytic modulation can fine-tune the balance of cell activation and inhibition (30
) and presumably also influence NK cell education. Intracellular trafficking pathways that contribute to expression of the less polymorphic NK cell receptors have been described. For the non-polymorphic activating receptor CD94/NKG2C and its inhibitory homologue CD94/NKG2A, separate pathways of endocytosis and recycling serve to maintain constant levels of unligated receptor at the cell surface (66
). Thus regulation of the secretory pathway for these receptors may have considerable influence on receptor expression levels. This is certainly the case for the activating receptors NKG2D, CD16, CD94/NKG2C, KIR3DS1 and KIR2DS in humans, as well as Ly49H and Ly49D in mice, whose cell surface expression is influenced by co-expression with a signaling adaptor protein (for example DAP12, DAP10, CD3ζ, or FcεR1γ) (31
The present study is the first to characterize the secretory pathway for inhibitory KIRs. KIR3DL1*004 represents an interesting example of how receptor polymorphism can influence NK cell function by affecting membrane traffic of the receptor. Our recent mutagenesis studies (28
) and the mapping of KIR3DL1*004 intracellular retention to the influence of only two residues, leucine 86 in the D0 domain and serine 182 in the D1 domain (27
), as well as earlier studies (71
), suggest that the conformation of KIR3DL1 receptors is very sensitive to sequence variation. We demonstrate here that the unfolded form of KIR3DL1*004 is more strongly associated with calreticulin than the readily expressed KIR3DL1*002 receptor. This raises the possibility that other KIR3DL1 allotypes with variable levels of cell surface expression (19
) reflect variable degrees of retention by calreticulin due to polymorphisms that affect their rate of folding. Our data suggest that expression of KIR3DL1*019, which only differs from KIR3DL1*004 at position 30, is regulated similarly. Several unfolded variants of KIR2DL2 and KIR2DS receptors have also been described (70
). It would now be of interest to determine whether these are truly null alleles for receptor expression or whether, as we have shown for KIR3DL*004, there are small amounts of these receptors that can fold properly for transport to the cell surface where they might function.
Our results demonstrate that intracellular KIR3DL1*004 is not folded properly and accumulates in the ER. This unfolded form of KIR3DL1*004 does not appear to function inside the cell, either by causing a cellular stress response as measured by chaperone synthesis or by induction of the UPR, or by influencing ligand expression and maturation. Very little co-immunoprecipitation of either KIR3DL1*004 or KIR3DL1*002 with HLA Class I ligand expressed in the same cell was detected. This trace interaction could arise from binding of solubilized ligands and receptors after cell lysis or from a very low level of intracellular co-association, which could occur between opposing intracellular membranes in a compartment or in a cis
-arrangement in the same membrane. In any case, the intracellular association of either KIR allotype with MHC class I is nowhere near as extensive as reported for Ly49 and its cognate ligand, which is readily detected (37
). Thus, our data suggest that KIR3DL1 function is confined to inhibitory signals transmitted by properly folded molecules at the cell surface upon interaction with their putative HLA-Bw4 ligand in trans
, on the surface of other cells. The unexpected finding that the low amount of KIR3DL1*004 expressed on the cell surface is functional, leads to the hypothesis that folding variation is a way to modulate receptor function. Consequently, allotypes with polymorphisms that affect folding are subject to the same selection for evolutionary advantage as other KIR allotypes.