The mechanisms by which the endosomal lumen triggers lipoprotein release from the LDLR are not fully understood. In in vitro
assays, both acidic pH and low calcium are sufficient to drive lipoprotein release. The acid-dependent mechanism requires the EGF-homology domain (20
), which forms an intramolecular contact with the ligand-binding domain at acidic pH (23
). This association appears to drive an allosteric process that accelerates lipoprotein release (24
). In cells, the acidification of endosomes also promotes the gating of an endosomal calcium channel, most likely the transient receptor potential V2 channel (33
). Activation of the endosomal calcium channel rapidly lowers the free calcium concentration in the endosomal lumen to micromolar concentrations (33
). Low free calcium can drive lipoprotein release by extracting calcium from the LA repeats and thereby inducing structural changes in the lipoprotein-binding surfaces on the LDLR (27
). Here, we show that this calcium-dependent mechanism accelerates release of both LDL and β-VLDL with rates similar to those of the acid-dependent mechanism (). We propose that both the acid-dependent and calcium-dependent mechanisms operate during endosomal release of lipoproteins. In the case of acid-dependent release, low calcium may act subsequent to lipoprotein dissociation to disrupt the closed conformational state of the LDLR (), thereby restoring the LDLR to an open conformation prior to delivery to the cell surface.
If both the acid-dependent and calcium-dependent processes provide efficient mechanisms of lipoprotein release, why have both? One potential reason for two mechanisms is to make the release process less reversible. The in vitro release experiments mimic three aspects of endosomal conditions: temperature (37°C), acidity (pH 5.5–6.0) and free calcium (pCa 5.0–5.6). One feature, which cannot be mimicked, is lipoprotein concentration. In the in vitro assays, lipoproteins release into the extracellular milieu, which has a large volume. As a consequence, release is irreversible because the concentration of lipoprotein is very low in the release media. By contrast, the endosomal lumen has a small volume and the endosomal concentration of lipoprotein is high after release. This high concentration of lipoprotein has the potential to reverse the release process. The combined action of the acid-dependent and calcium-dependent mechanisms may prevent rebinding of lipoproteins after release.
The inability of LDLR-ΔBC cells to support LDL uptake indicates that the calcium-dependent process is not sufficient for cellular release of LDL and that LDL uptake requires the acid-dependent release process. Consistent with this observation, mutations that increase the acid requirement for acid-dependent release impair cellular uptake of LDL (24
). Interestingly, mutations that increase the acid requirement by as little as 0.5 pH units can reduce LDL uptake by more than 90%, suggesting that LDL uptake is exquisitely sensitive to the acid-dependent release process.
Whether LDL uptake also requires the calcium-dependent process is not clear. The LDLR completes a cycle of LDL uptake every ~12 min (49
), indicating that LDL release occurs in early endosomes, where the pH is ≥6.0 and free calcium concentrations are ≥10 μM (33
). Comparison of the calcium-dependent rate constant of LDL release by LDLR-ΔBC at pH 6.0 and pCa 5.0 (9.5×10−4
) with acid-dependent release by WT LDLR at pH 6.0 and pCa 2.7 (1.9×10−3
) indicates that the acid-dependent process is a stronger driver of LDL release at early endosomal conditions (). However, LDL release by WT LDLR at pCa 5.0 (3.1×10−3
) was faster than at pCa 2.7 (1.9×10−3
), suggesting that low free calcium may facilitate LDL release. Unfortunately, inhibition of the endosomal calcium channel does not provide a means to determine whether LDL uptake requires the calcium-dependent process because fusion of primary endosomes with early endosomes requires calcium efflux from the endosomal lumen (50
). Use of the calcium ionophore, A23187, to increase cellular calcium and collapse the endosomal calcium gradient inhibits LDL uptake by ~60% (53
). This observation is consistent with the possibility that LDL release is inhibited when endosomal calcium cannot be reduced; however, calcium plays multiple roles in endocytosis and elevated levels of intracellular calcium can promote, inhibit or have little effect on endocytic uptake depending upon the ligand and system (54
A key observation of this study is that β-VLDL uptake is normal in LDLR-ΔBC expressing cells. This observation indicates that the acid-dependent lipoprotein release is not required for β-VLDL uptake and suggests that the calcium-dependent process may be the principal driver of β-VLDL release. Two questions raised by this observation are first, why does β-VLDL uptake not require acid-dependent release and second, why is the process of endosomal release of β-VLDL not sufficient for LDL release?
One potential answer to the question of why β-VLDL uptake does not employ the acid-dependent lipoprotein release is the observation that acid-dependent β-VLDL release requires harsher acidic conditions than LDL release ( and ). This difference in pH sensitivity may be caused by the higher affinity of the LDLR for β-VLDL as compared to LDL or by the ability of multiple LDLR to bind β-VLDL simultaneously as compared to the 1:1 stoichiometry between LDL and the LDLR (58
). Consistent with the latter possibility, the number of apoE present on β-VLDL influences uptake of β-VLDL (60
). Reduced sensitivity to low pH may thus preclude the acid-dependent mechanism for cellular release of β-VLDL.
A likely key to the question of why the mechanism driving β-VLDL release cannot support LDL release is differences in the intracellular trafficking of LDL and β-VLDL after internalization. Comparison of the endocytic path of LDL and β-VLDL in macrophages has shown that while internalized LDL rapidly accumulates in late endosomes and lysosomes, internalized β-VLDL accumulates first in peripheral structures, presumably an endosome-like compartment, before trafficking to degradative compartments (60
). This bypass suggests that β-VLDL may be held in a specialized endocytic compartment to provide the LDLR-β-VLDL complex with additional time or a more conducive environment for release. Targeting of β-VLDL to these compartments appears to involve engagement of multiple LDLR because accumulation of β-VLDL in these peripheral structures is augmented by increased apoE content of β-VLDL (60
). While similar experiments have not been done in fibroblasts, fibroblasts show delayed degradation of apoE containing lipoproteins (61
), consistent with the possibility that fibroblasts also have a bypass segment in the degradative pathway of β-VLDL. Residence of β-VLDL in peripheral endosome-like structures may facilitate the calcium-dependent release process.
The ability of the LDLR-ΔBC, but not LDLR-ΔAC, cells to support β-VLDL uptake suggests that the EGF-A module plays a critical role in receptor trafficking. Consistent with this possibility, natural deletions that remove either both EGF-A and EGF-B or just EGF-A result in familial hypercholesterolemia (FH) (65
) and fibroblasts from individuals that are homozygous for the deletion removing both the EGF-A and EGF-B modules express an LDLR variant that recycles poorly in the presence of β-VLDL (70
). The EGF-A module is also been implicated in LDLR trafficking because the secreted protein, PCSK9, binds to EGF-A and promotes LDLR degradation by redirecting internalized LDLR to lysosomes (71
). The observation that the LDLR-ΔBC receptor was less sensitive than the LDLR-ΔAC to monensin () also suggests that the EGF-A promotes receptor recycling. Interestingly, the WT LDLR is sensitive to monensin and this sensitivity is heightened in the presence of lipoprotein (42
). The reduced sensitivity of the LDLR-ΔBC to monensin/lipoprotein co-treatment indicates that the EGF-B to EGF-C (BC) region enhances the monensin sensitivity of the WT LDLR and suggests that the BC region may act to inhibit recycling prior to lipoprotein release. The process of recycling prior to lipoprotein release is termed retro-endocytosis and in fibroblasts, accounts for ~10% of the LDL that is internalized by the LDLR (72
). Interestingly, LDLR-ΔBC cells bind LDL normally; however, these cells are unable to accumulate LDL and the surface level of receptors does not change in the presence of LDL (, and ). These observations suggest that the LDLR-ΔBC recycles prior to lipoprotein release. The BC region may thus not only participate in acid-dependent release, but may also prevent receptor recycling until release has occurred.
In summary, both acidic pH and low free calcium can drive lipoprotein release. The acid- dependent process is required for LDL uptake, but not for β-VLDL uptake, suggesting that the calcium-dependent process may participate in the cellular release of β-VLDL. Cellular accumulation of β-VLDL requires the EGF-A module, which appears to participate in LDLR trafficking.