Our findings combined with previous data demonstrate that HLA-F is expressed independent of bound peptide, at least with respect to peptide complexity profiles similar to those of either HLA-E or classical MHC-I. In addition, we were able to demonstrate that not only was HLA-F surface expression coincident with MHC-I HC expression, but that HLA-F surface expression was down regulated upon perturbation of MHC-I HC structure. It was further possible to directly demonstrate that MHC-I would only interact with HLA-F in the form of open conformer probably free of peptide and not as peptide bound complex. This interaction was directly observed intracellularly through co-immunoprecipitation and surface plasmon resonance and indirectly on the surface of cells through coincident tetramer and MHC-I HC co-localization. Together these data suggest that HLA-F is expressed independent of peptide and that a physical interaction specific to MHC-I HC plays a role in the functional consequence of MHC-I HC expression in activated lymphocytes.
Although our results suggest that HLA-F is expressed as HC without peptide, immunoprecipitations using our panel of HLA-F specific antibodies do coprecipitate β2
m in subequimolar amounts (23
). Based on this data and the differential coimmunoprecipitation experiments reported here (), it appears likely that more than one complex form of HLA-F is expressed, with a portion complexed with β2
m, and some or all complexed with MHC-I HC. In total there is evidence of at least three different forms of HLA-F based on differential staining of surface HLA-F using antibodies 4A11 and 3D11 over the course of lymphocyte activation and the unique 4B4 binding pattern. In light of this, it is also possible that HLA-F may bind larger peptides, beyond the limits of our analysis of 13mers, or may be stabilized by interactions with proteins other than MHC class I or other biomolecules such as phospholipids similar to CD1d (53
). Interestingly, the HLA-F protein is entirely dependent on its cytoplasmic tail for export from the ER and the HLA-F cytoplasmic sequence contains motifs that are overlapping with those of CD1d (24
It is possible to refold HLA-F with β2
m without peptide which forms a stable structure that can be used in tetramer binding ((55
) and ). Open conformer MHC-I are relatively unstable in comparison but are up-regulated by cold treatment, while HLA-F is not, suggesting that there is likely a threshold of stability that MHC class I HC must reach to traffic to and remain on the cell surface. The possibility that HLA-F is required for the formation of the open conformer is raised considering their coincident surface expression and physical interaction. Since HLA-F is otherwise expressed intracellularly in resting lymphocytes it is also possible that the formation of MHC-I HC upon activation induces complex formation between HLA-F and MHC-I HC, which then signals transport to the surface.
When free HC antibodies bind to MHC-I HC on the cell surface, the conformation of the HC may be changed to more closely resemble MHC complex with peptide. Whether HLA-F recycles with MHC-I HC as suggested by internalization upon addition of HC antibodies would clearly add an important dimension to studies of the trafficking of MHC-I HC. As an interesting supplement, HLA-F does have a tyrosine based internalization motif (TSQA) similar to that proposed to regulate MHC-I endocytosis and intracellular trafficking, while HLA-C an HLA-G do not (56
). This may be relevant to HLA-C and HLA-G cycling in trophoblast cells in light of the coincident expression of these molecules in conjunction with HLA-F in the placental environment (12
). It is not known whether HLA-C is expressed as a free heavy chain in trophoblasts, but evidently HLA-G is (15
) and in tropohoblasts that have invaded the maternal decidua, HLA-F and HLA-G are co-expressed on the surface. It would be interesting to explore the idea that the lack of the internalization motif on HLA-C and HLA-G was directly related to their expression in the placental environment.
Our data do not address whether HLA-F interacts with all MHC-I HC since we were able to analyze only those MHC-I for which a conditional ligand was available to us (HLA-A2 and -A3) or that we were able to refold without peptide (HLA-E). Considering the extensive polymorphism of MHC class I, it is possible that HLA-F interacts with only a subset of MHC-I alleles or more generally stated that the affinity between HLA-F and different MHC-I alleles will differ substantially. Our analysis of the three MHC-I HC studied in this report was not carried out under conditions that allowed for precise quantitative measurement, however differences could be detected between HLA-A2 or HLA-A3 and HLA-E. While we do not have detail on the interaction points between HLA-F and MHC-I HC, a logical presumption from this work would be that they are restricted to regions exposed upon removal of peptide.
The reactivities of MHC-I HC specific mAbs has been mapped to epitopes formed by residues within positions 57-84 in the a1 domain that are specific to the open conformer (10
). These residues are in contact with peptide or otherwise hidden in the peptide bound molecule but become exposed in the MHC-I HC conformer. HCA2 has been mapped to residues 77-84 probably ruling this segment out since HLA-F and HCA2 do not compete for binding to MHC-I, but a similar comparative analysis with other MHC-I HC specific antibodies could be useful in mapping the HLA-F contact residues. Further insight may come from comparative sequence analysis with HLA-F homologues expressed in other primates. For example, HLA-F homologues are found in macaques with as few as 10 amino acid differences in the α1 to α3 domains (26
These findings leave open the possibility that HLA-F binds to a specific receptor(s) or even that HLA-F and MHC-I HC interactions can occur in trans between cells. Addressing the former possibility, while our immunoprecipitations with 3D11 and 4A11 did not co-precipitate equimolar amounts of MHC-I as did 4B4, it is unlikely that this evidence argues against an interaction of HLA-F and MHC-I HC on the surface. Subequimolar amounts were detected with both antibodies and it is not clear that this was due to experimental conditions (e.g. the detergent used) and possibly compounded by antibody specificities. In fact, there is a strong correlation with binding of HLA-F tetramer and the presence of surface MHC-I HC (). In addition, surface HLA-F is internalized upon addition of free HC antibodies, which could implicate an interaction between the two molecules prior to the addition of HC antibody. If heterodimers of MHC-I HC and HLA-F or similar complexes of homodimers of each species do exist on the surface. it would suggest the possibility for interaction with a unique receptor in trans, perhaps another polymorphic locus or group of genes.
MHC-I interactions with other proteins have been demonstrated in cis
, including insulin and epidermal growth factor receptors, and evidence points to that interaction being exclusively with MHC-I HC (57
). Indeed a relatively long list of reported cis-associations between MHC-I and other surface receptors and proteins can be compiled from the literature (10
). Whether all of these associations are formed between the MHC-I open conformer remains to be determined, but the discovery we report here may place a new light on these interactions and indeed may refocus some emphasis on this aspect of MHC-I function.
Further functional data will clarify the role that interaction between HLA-F and MHC-I plays in class I presentation by activated cells. However, the high conservation of HLA-F and the binding to MHC-I HC together suggest a role as chaperone to stabilize MHC-I HC expression in the absence of peptide. This escort function could be operating in both directions, bringing MHC-I HC to the surface and internalizing MHC-I HC after appropriate signals have been delivered, although we have no direct evidence in support of the latter possibility. The coincident internalization of HLA-F may therefore suggest an involvement in internalization of MHC-I upon encounter with the formation of an alternative stable complex.