Portal de Periódicos da CAPES

The Switch on the RAPper's Necklace…

2006; Elsevier BV; Volume: 23; Issue: 4 Linguagem: Inglês

10.1016/j.molcel.2006.07.026

1097-4164

Joachim Herz,

Protein Structure and Dynamics

The biosynthesis and export of LDL receptor-related proteins rely on specialized chaperones in the endoplasmic reticulum. Two recent papers in Molecular Cell by Fisher et al., 2006Fisher C. Beglova N. Blacklow S.C. Mol. Cell. 2006; 22: 277-283Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar and Lee et al., 2006Lee D. Walsh J.D. Mikhailenko I. Yu P. Migliorini M. Wu Y. Krueger S. Curtis J.E. Harris B. Lockett S. et al.Mol. Cell. 2006; 22: 423-430Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar reveal a novel mechanism by which one of these chaperones, the receptor-associated protein RAP, accomplishes this task. The biosynthesis and export of LDL receptor-related proteins rely on specialized chaperones in the endoplasmic reticulum. Two recent papers in Molecular Cell by Fisher et al., 2006Fisher C. Beglova N. Blacklow S.C. Mol. Cell. 2006; 22: 277-283Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar and Lee et al., 2006Lee D. Walsh J.D. Mikhailenko I. Yu P. Migliorini M. Wu Y. Krueger S. Curtis J.E. Harris B. Lockett S. et al.Mol. Cell. 2006; 22: 423-430Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar reveal a novel mechanism by which one of these chaperones, the receptor-associated protein RAP, accomplishes this task. Molecular chaperones are a structurally and functionally diverse class of proteins that come in many shapes and sizes. As the name indicates, their primary role is to protect or support the functions of other proteins. Some chaperones have broad specificity, inasmuch as they associate with a large number of target proteins; others are highly specialized and tailored to the needs of a single protein or molecular process. They are ubiquitous, from simple prokaryotes to mammals, and, as a whole, they are essential to cellular survival. Major functions of chaperones are, for instance, to assist in protein folding, in quality control, as escort proteins in the secretory pathway, or in the temporary “functional occlusion” of the target protein. The cytosolic heat shock proteins, the ER chaperones calnexin and grp78, and the invariant chain that protects MHC class II molecules from becoming occupied by cellular autoantigens in the secretory pathway before they reach the cell surface are just a few of the best-known examples. Their functions are crucial to ensure proper protein folding, to respond to and protect the cell from environmental stress, and to resolve the conflicts that arise during the synthesis of two proteins (or independently folded parts of the same protein) that interact with high affinity in the same cell. A specialized chaperone that serves both as an escort protein and as a temporary blocker of target protein function is the receptor-associated protein RAP. At first considered merely a “pesky contaminant” during the purification of the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) (Ashcom et al., 1990Ashcom J.D. Tiller S.E. Dickerson K. Cravens J.L. Argraves W.S. Strickland D.K. J. Cell Biol. 1990; 110: 1041-1048Crossref PubMed Scopus (203) Google Scholar, Strickland et al., 1990Strickland D.K. Ashcom J.D. Williams S. Burgess W.H. Migliorini M. Argraves W.S. J. Biol. Chem. 1990; 265: 17401-17404Abstract Full Text PDF PubMed Google Scholar), it has since morphed into a role in which it controls the biosynthesis of this multifunctional receptor, and also into a versatile tool that has greatly facilitated the study of the biological functions of the entire LDL receptor gene family. This evolutionarily ancient family comprises seven multifunctional cell surface receptors in mammalian species, i.e., the LDL receptor, very-low-density lipoprotein receptor (VLDLR), Apolipoprotein E receptor-2 (ApoER2), LRP1, LRP1b, Megalin, and MEGF7/LRP4. The family evolved along with the first metazoans, and the basic structural organization has remained virtually unchanged throughout the course of evolution (reviewed in Willnow et al., 1999Willnow T.E. Nykjaer A. Herz J. Nat. Cell Biol. 1999; 1: E157-E162Crossref PubMed Scopus (186) Google Scholar). The different family members control highly diverse and seemingly unrelated physiological processes, including lipoprotein metabolism, cholesterol transport, protease and vitamin metabolism, smooth muscle proliferation and vascular wall integrity, neuronal migration during brain development, synaptic neurotransmission, and limb patterning. Aside from cargo transport functions, such as the removal of cholesterol and lipoproteins from the circulation, several family members such as LRP1, VLDLR, ApoER2, and MEGF7/LRP4 can either transmit extracellular signals directly or they can modulate other signal transduction pathways, e.g., through PDGF, BMPs, or WNTs. The myriad of proteins that can bind to or otherwise interact with the different family members places these multifunctional sensors of the extracellular environment into a unique position from which they can control or modulate cellular responses to signal input through these other pathways. In light of the broad physiological functions of the LDL receptor gene family and the fundamental role RAP plays in controlling the biosynthesis and export of the receptors, a comprehensive understanding of the underlying molecular mechanism is crucial. Recent studies by Lee et al., 2006Lee D. Walsh J.D. Mikhailenko I. Yu P. Migliorini M. Wu Y. Krueger S. Curtis J.E. Harris B. Lockett S. et al.Mol. Cell. 2006; 22: 423-430Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar and Fisher et al., 2006Fisher C. Beglova N. Blacklow S.C. Mol. Cell. 2006; 22: 277-283Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar now provide complementary structural insights into the atomic interactions through which RAP regulates this process. This review attempts to put these novel findings and their significance for the physiological functions of the LDL receptor gene family into a common perspective. When RAP was first identified as a small protein that copurified with LRP1 (a.k.a. the α2-macroglobulin receptor), its functional significance was not at all clear (Ashcom et al., 1990Ashcom J.D. Tiller S.E. Dickerson K. Cravens J.L. Argraves W.S. Strickland D.K. J. Cell Biol. 1990; 110: 1041-1048Crossref PubMed Scopus (203) Google Scholar, Strickland et al., 1990Strickland D.K. Ashcom J.D. Williams S. Burgess W.H. Migliorini M. Argraves W.S. J. Biol. Chem. 1990; 265: 17401-17404Abstract Full Text PDF PubMed Google Scholar). RAP was not required for ligand binding; on the contrary, purified or recombinantly produced RAP strongly antagonized the binding of all the LRP1 ligands that were known at the time (Herz et al., 1991Herz J. Goldstein J.L. Strickland D.K. Ho Y.K. Brown M.S. J. Biol. Chem. 1991; 266: 21232-21238Abstract Full Text PDF PubMed Google Scholar, Williams et al., 1992Williams S.E. Ashcom J.D. Argraves W.S. Strickland D.K. J. Biol. Chem. 1992; 267: 9035-9040Abstract Full Text PDF PubMed Google Scholar). This property as a universal inhibitor of ligand binding to LDL receptor-related proteins has made recombinant RAP a widely used tool for the study of the biochemical properties of the LDL receptor gene family. The same property is also essential for the physiological role of RAP as an ER chaperone, which was revealed through a combination of cell biological and genetic approaches. Bu and colleagues (Bu et al., 1995Bu G. Geuze H.J. Strous G.J. Schwartz A.L. EMBO J. 1995; 14: 2269-2280Crossref PubMed Scopus (270) Google Scholar) showed that the carboxy-terminal HNEL tetraamino acid sequence is necessary and sufficient for RAP retention in the ER. They also showed that RAP only transiently associates with LRP1 and that it dissociates from the receptor after it enters later compartments of the secretory pathway with a lower pH, consistent with its requirement for neutral pH and [Ca2+] for binding to LRP1 (Herz et al., 1991Herz J. Goldstein J.L. Strickland D.K. Ho Y.K. Brown M.S. J. Biol. Chem. 1991; 266: 21232-21238Abstract Full Text PDF PubMed Google Scholar). This dissociation of RAP from LRP1 correlated with an increase in receptor ligand binding activity. In another study, a reduction in LRP1, but not LDL receptor expression levels, was found in RAP knockout mice, indicating that RAP selectively controls the activity of LDL receptor family members in vivo. The findings by Fisher et al., 2006Fisher C. Beglova N. Blacklow S.C. Mol. Cell. 2006; 22: 277-283Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar and Lee et al., 2006Lee D. Walsh J.D. Mikhailenko I. Yu P. Migliorini M. Wu Y. Krueger S. Curtis J.E. Harris B. Lockett S. et al.Mol. Cell. 2006; 22: 423-430Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar now show not only how RAP prevents ligand binding through a mechanism that may generically apply to many of the ligand binding domains in the LDL receptor family with which RAP has been shown to interact, they also reveal a novel histidine switch mechanism by which RAP dissociates from LRP1 under the mildly acidic and low [Ca2+] conditions that prevail in early Golgi compartments. As a soluble ER protein of approximately 40 kDa, RAP interacts with high affinity with the ligand binding domains of most members of the LDL receptor gene family (the LDL receptor being a notable and significant exception, as discussed below) as they emerge from the lumenal ER membrane, undergo folding, and acquire the ability to recognize ligands. This binding of RAP to the newly folded receptor ligand binding domains is crucial to prevent the interaction of the nascent and structurally immature receptors with their cognate ligands that are often synthesized simultaneously in the same compartment. The presence of high local concentrations of receptors and ligands in a confined compartment, as they are present in the ER, shifts the equilibrium toward conditions that favor premature binding of these ligands to their receptors. If this happens before the receptor, the ligand, or both have completed biosynthesis and folding, they can aggregate into large insoluble complexes that may chronically trigger unfolded protein responses, impair ER function, interfere with secretion, or, at a minimum, reduce biosynthetic efficiency. Thus, RAP can be considered as a “lubricant” that reduces “friction” and facilitates the smooth export of the receptors from the ER (Figure 1). Normally, the concentrations of RAP in the ER are sufficient to prevent such premature interactions and aggregation. However, under certain experimental conditions, such aggregates can be induced. For instance, adenoviral overexpression of Apolipoprotein E, a ligand for all members of the family, resulted in the aggregation of LRP1 in the ER. This occurred even in the presence of endogenously produced RAP, suggesting that the high levels of ApoE outcompeted RAP for binding to LRP1. Coexpression of RAP restored normal endogenous receptor expression (Willnow et al., 1996Willnow T.E. Rohlmann A. Horton J. Otani H. Braun J.R. Hammer R.E. Herz J. EMBO J. 1996; 15: 2632-2639Crossref PubMed Scopus (237) Google Scholar). Complementary results have been achieved in converse experiments in which an entire receptor or ligand binding domains were overexpressed, resulting in the relative depletion of RAP. Simultaneous expression of RAP restored normal export of the receptors from the ER (Lee et al., 2006Lee D. Walsh J.D. Mikhailenko I. Yu P. Migliorini M. Wu Y. Krueger S. Curtis J.E. Harris B. Lockett S. et al.Mol. Cell. 2006; 22: 423-430Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, Obermoeller-McCormick et al., 2001Obermoeller-McCormick L.M. Li Y. Osaka H. FitzGerald D.J. Schwartz A.L. Bu G. J. Cell Sci. 2001; 114: 899-908Crossref PubMed Google Scholar), suggesting that the relative concentrations of RAP, receptors, and coexpressed ligands are balanced to fit the needs of the individual cell type or tissue. Gene knockout experiments of RAP showed that in some tissues, such as the liver or neurons, RAP plays a more prominent role in ensuring normal receptor biosynthesis than in others. A likely reason for this is the different levels at which ligands for one receptor or another are expressed in the same cell. For instance, LRP1 is synthesized normally in RAP-deficient fibroblasts, whereas functional expression of LRP1, but not of the LDL receptor, is reduced by approximately 80% in the livers and brains of RAP knockout mice (Willnow et al., 1996Willnow T.E. Rohlmann A. Horton J. Otani H. Braun J.R. Hammer R.E. Herz J. EMBO J. 1996; 15: 2632-2639Crossref PubMed Scopus (237) Google Scholar). Although there is no direct evidence that regulated changes in RAP expression occur in vivo and that this would bear any physiological significance on the function of LDL receptor-related proteins, there is indirect evidence to suggest that the LDL receptor may have selectively evolved to express only weak affinity for RAP. Biosynthetic studies on Apolipoprotein B, the protein that mediates the binding of LDL to the LDL receptor, showed that the interaction of nascent lipoprotein particles with the LDL receptor in the secretory pathway significantly affected the secretion of endogenous lipoproteins from the liver (Twisk et al., 2000Twisk J. Gillian-Daniel D.L. Tebon A. Wang L. Barrett P.H. Attie A.D. J. Clin. Invest. 2000; 105: 521-532Crossref PubMed Google Scholar). Thus, the LDL receptor can physiologically control plasma cholesterol levels not only through the endocytosis of LDL, but also already on the presecretory level, by directly shunting nascent lipoprotein particles in the secretory pathway toward degradation. Any higher affinity of RAP for the LDL receptor might abrogate this regulatory mechanism, resulting in a higher lipoprotein secretion rate and elevated, atherosclerosis-promoting plasma LDL levels. The results of adenoviral in vivo gene transfer and overexpression of RAP in mice, which led to the accumulation of VLDL and LDL cholesterol in the circulation (Willnow et al., 1994Willnow T.E. Sheng Z. Ishibashi S. Herz J. Science. 1994; 264: 1471-1474Crossref PubMed Scopus (255) Google Scholar), support such a model. The novel insights into the three-dimensional structure of RAP have now led to a clearer understanding of the conformational changes and charge redistributions that occur at the surface of the protein when it enters a low pH environment in the secretory pathway. The atomic interactions RAP forms with the backbone of the receptor ligand binding domains further explain how RAP can universally inhibit the binding of such a wide spectrum of ligands across the different family members. Fisher et al., 2006Fisher C. Beglova N. Blacklow S.C. Mol. Cell. 2006; 22: 277-283Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar and Lee et al., 2006Lee D. Walsh J.D. Mikhailenko I. Yu P. Migliorini M. Wu Y. Krueger S. Curtis J.E. Harris B. Lockett S. et al.Mol. Cell. 2006; 22: 423-430Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar used different and complementary methods, X-ray crystallography and NMR spectroscopy, respectively, to provide us with a comprehensive understanding of these mechanisms. RAP is made up of three helical domains of roughly 100 amino acids each. In their studies, Fisher et al., 2006Fisher C. Beglova N. Blacklow S.C. Mol. Cell. 2006; 22: 277-283Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar extended earlier groundbreaking work on the structure of LDL receptor ligand binding type A (LA) modules from their own laboratory (Fass et al., 1997Fass D. Blacklow S. Kim P.S. Berger J.M. Nature. 1997; 388: 691-693Crossref PubMed Scopus (302) Google Scholar) to generate and analyze cocrystals of two LA modules with the high-affinity receptor-interacting domain 3 of RAP (RAP-D3). For technical reasons that had to do with crystal growth and diffraction conditions, they chose a single LA module pair (3–4) of the LDL receptor, which is the only pair in the LDLR that probably binds to the RAP D3 domain with an affinity comparable to that generally observed for most LRP1 module pairs to obtain a 1.26 Å resolution structure. The results were highly revealing. The same coordinated Ca2+ ions that are crucial for folding, as well as for maintaining the structural integrity of the LA modules, are also critical for the proper presentation of the two aspartate side chains that are coordinated by the ion in each module to conserved lysine residues within the three-helix bundle of the RAP-D3 domain. The authors fittingly call this unique structure an “acidic necklace” that surrounds the neck of the lysine side chain. Since the contact area between the RAP-D3 domain and each individual module is quite small, this simultaneous but discontinuous two-point interaction of the RAP-D3 domain with two modules is critical for attaining high-affinity interaction of RAP with the ligand binding domains. The importance of these interactions, and specifically the role of the lysine residues in the RAP-D3 domain that form the contact with the acidic necklaces in the LA modules, is underscored by earlier random mutagenesis studies of the RAP-D3 domain (Migliorini et al., 2003Migliorini M.M. Behre E.H. Brew S. Ingham K.C. Strickland D.K. J. Biol. Chem. 2003; 278: 17986-17992Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) that found only two residues in this domain that were critical for binding to LRP1—the very same lysines that are now found to “wear” those necklaces. It should be noted that the D3 domain is not solely responsible for achieving the full affinity of RAP for receptor binding but that the D1 and D2 domains also contribute, albeit to a smaller extent. There is compelling evidence to suggest that the mode of interaction Fisher et al., 2006Fisher C. Beglova N. Blacklow S.C. Mol. Cell. 2006; 22: 277-283Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar have solved in their cocrystallization study will generally hold true for the interaction of RAP as well as other ligands with LA modules. First, a number of proteins that bind to LDL receptor family members are known to do so through interactions that involve clusters of basic residues. Furthermore, the residues that form the necklace around the lysine side chains have been shown to be critical for ligand binding to LA modules (Andersen et al., 2000Andersen O.M. Christensen L.L. Christensen P.A. Sorensen E.S. Jacobsen C. Moestrup S.K. Etzerodt M. Thogersen H.C. J. Biol. Chem. 2000; 275: 21017-21024Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Moreover, proper Ca2+ coordination by these residues is critical for forming the necklace as well as for ligand binding. Additional convincing and independent evidence for the importance of the “lysine necklace” as a binding mechanism comes from the structure that has been solved for the LDL receptor at endosomal, i.e., acidic pH (Rudenko et al., 2002Rudenko G. Henry L. Henderson K. Ichtchenko K. Brown M.S. Goldstein J.L. Deisenhofer J. Science. 2002; 298: 2353-2358Crossref PubMed Scopus (376) Google Scholar). Under these conditions, ligands are unable to bind, because the aspartates of LA modules 4 and 5 form high-affinity necklace interactions with analogous lysine residues within the YWTD propeller domain. Fisher et al., 2006Fisher C. Beglova N. Blacklow S.C. Mol. Cell. 2006; 22: 277-283Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar have taken the information they obtained from their crystal structure a step further, and on this basis they have modeled the likely interaction of a biomedically important LDL receptor ligand, ApoE, with the LA modules. Strikingly, one of the critical lysines that mediate ApoE binding to the LDL receptor (K146) matches almost perfectly with one of the “necklace-wearing” lysines (K256) in the RAP-D3 domain when the RAP and ApoE structures are superimposed. Another basic residue in ApoE that is important for receptor binding (R172) is repositioned upon interaction of ApoE with lipid, and under these conditions it forms an auxiliary docking site with the backbone of the ligand binding domain. Furthermore, the micellar structure of lipid particles allows for the presence of more than one ApoE moiety per particle, thereby increasing avidity through additional interaction sites, similar to the two lysine necklaces that are required for high-affinity RAP binding. The findings of Fisher et al., 2006Fisher C. Beglova N. Blacklow S.C. Mol. Cell. 2006; 22: 277-283Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar comprehensively reveal the molecular contact sites through which RAP binds to LDL receptor family members and thereby show how these interactions can occupy and thus block the receptor ligand binding domains. Taken together with the results reported by Lee et al., 2006Lee D. Walsh J.D. Mikhailenko I. Yu P. Migliorini M. Wu Y. Krueger S. Curtis J.E. Harris B. Lockett S. et al.Mol. Cell. 2006; 22: 423-430Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar both reports now also provide deep and novel insights into the mechanisms that underlie the role of RAP as an escort protein. By determining the NMR solution structure of the RAP-D3 domain, Lee and colleagues were intrigued by a striking pH-dependent signature shift in the electrostatic surface potentials of this domain, which involves a series of exposed histidines. Both groups further draw attention to the potential role of the buried histidine residues in promoting a pH-dependent unfolding of the D3 domain. These findings point to a novel histidine switch mechanism that mediates the catch-and-release through which RAP lets go of the receptors as soon as they enter the more acidic Golgi compartments. Histidines have a near-neutral pKa value, which means in practical terms that the charge carried by these side chains will flip during the passage of the protein from the ER to the Golgi. Lee et al., 2006Lee D. Walsh J.D. Mikhailenko I. Yu P. Migliorini M. Wu Y. Krueger S. Curtis J.E. Harris B. Lockett S. et al.Mol. Cell. 2006; 22: 423-430Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar show that this charge reversal results in a global change of the electrostatic surface potential of the D3 domain, which is accompanied by a reversible conformational change of the entire domain. Although none of the histidine residues are directly involved in LA module interaction (Fisher et al., 2006Fisher C. Beglova N. Blacklow S.C. Mol. Cell. 2006; 22: 277-283Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), two of these residues are close enough to potentially influence the stability of the necklace from a distance. However, alanine replacement of either of these histidines in isolation did not affect RAP binding to LRP1 at neutral or acidic pH, suggesting that either both of these residues must be replaced to noticeably affect binding, that the acid-induced unfolding is the primary mechanism that drives dissociation of RAP from the LA modules, or that both mechanisms cooperate in achieving this. To functionally demonstrate the significance of their proposed histidine switch mechanism, Lee et al., 2006Lee D. Walsh J.D. Mikhailenko I. Yu P. Migliorini M. Wu Y. Krueger S. Curtis J.E. Harris B. Lockett S. et al.Mol. Cell. 2006; 22: 423-430Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar employed a combination of biochemical in vitro analysis and cell culture experiments. They first generated mutant RAP proteins in which all the exposed, as well as the buried, histidine residues in domains D1, D2, D3, or all domains had been replaced by alanines. All of these constructs bound to LRP1, although D3 mutants had a slightly lower affinity. They next tested binding and dissociation properties of their mutant proteins at different pH conditions. Wild-type RAP readily dissociated from LRP1 already under mildly acidic conditions, while the D3 mutants were resistant to acid-induced dissociation. They then went on to investigate the effect of the histidine mutations on the trafficking of a soluble, i.e., nonmembrane anchored LRP minireceptor (sLRP) by monitoring its rate of secretion from transfected cells into the cell culture medium. Efficient secretion of the soluble minireceptor was dependent upon the coexpression of RAP, confirming earlier findings that the unphysiological overexpression of a receptor in the absence of sufficient amounts of RAP results in aggregation and ER retention. Overexpression of RAP promoted the secretion of the sLRP through the secretory pathway into the medium. RAP was secreted along with the minireceptor, presumably because the high expression levels overwhelmed the ER retention mechanisms (Willnow et al., 1994Willnow T.E. Sheng Z. Ishibashi S. Herz J. Science. 1994; 264: 1471-1474Crossref PubMed Scopus (255) Google Scholar). The D3 histidine mutants, by contrast, were less efficient in promoting sLRP secretion and, despite equivalent expression levels, far less RAP was found in the medium. FRET analysis confirmed that sLRP was preferentially retained in the ER in a complex with RAP and only small amounts were present in Golgi compartments. The most straightforward explanation for this finding is that sLRP is being shuttled back to the ER along with RAP when the two proteins fail to dissociate in the acidic Golgi compartments. However, this scenario cannot fully explain why, in contrast to wild-type RAP, the D3 histidine mutants failed to be efficiently secreted into the culture medium. This is especially puzzling, since sLRP was secreted under these conditions, albeit at a reduced amount. If the mutant RAP cannot dissociate from sLRP in the Golgi compartments, where did it go? Why was the ER retention mechanism not overwhelmed by the equally high expression levels of the mutant RAP? A possible partial explanation for these observations might be that the mutant RAP is being degraded along with the bound sLRP from which it cannot be released upon retrieval to the ER. During folding and maturation, secreted proteins routinely undergo repeated cycles of quality checks in which other ER chaperones such as calnexin and calreticulin cooperate with glucosidases to optimize their three-dimensional structure (reviewed in Ellgaard and Helenius, 2003Ellgaard L. Helenius A. Nat. Rev. Mol. Cell Biol. 2003; 4: 181-191Crossref PubMed Scopus (1672) Google Scholar). LDL receptor family members are particularly dependent on these quality checks, as they consist of two highly complex domains, the ligand binding and the YWTD propeller domain, which intramolecularly interact with each other (Rudenko et al., 2002Rudenko G. Henry L. Henderson K. Ichtchenko K. Brown M.S. Goldstein J.L. Deisenhofer J. Science. 2002; 298: 2353-2358Crossref PubMed Scopus (376) Google Scholar). Through the acidic necklace, RAP not only prevents premature ligand binding, it also prevents the premature interaction of the ligand binding domain with the YWTD propeller. It is possible that this interaction, if it occurs before folding of the receptors is complete, would prevent or delay their folding and maturation. In this capacity, RAP binding to the LA modules would ensure that the calnexin/calreticulin/glucosidase cycle, and thus the folding of the highly complex YWTD propeller domain involving Boca, another specialized chaperone (Culi et al., 2004Culi J. Springer T.A. Mann R.S. EMBO J. 2004; 23: 1372-1380Crossref PubMed Scopus (44) Google Scholar), can continue. On the other hand, failure of RAP to release the receptors might interfere with the continuing maturation of carbohydrate side chains (McCormick et al., 2005McCormick L.M. Urade R. Arakaki Y. Schwartz A.L. Bu G. Biochemistry. 2005; 44: 5794-5803Crossref PubMed Scopus (17) Google Scholar) and result in its degradation. Or, perhaps more likely, the inability to release a bound ligand and to fold the ligand binding domain back on the YWTD propeller under acidic conditions targets the receptor toward lysosomal degradation. This mechanism would be consistent with the findings of Davis et al., 1987Davis C.G. Goldstein J.L. Sudhof T.C. Anderson R.G. Russell D.W. Brown M.S. Nature. 1987; 326: 760-765Crossref PubMed Scopus (309) Google Scholar, who showed that an LDL receptor that lacks the YWTD propeller domain is being transported normally to the cell surface but fails to recycle and is rapidly degraded upon ligand binding and internalization. It would be informative to test whether lysosomotropic agents, such as chloroquine, prevent the degradation of the His mutant RAP/sLRP complex. In any event, the novel insights that have arisen from the elegant and complementary studies by Fisher et al., 2006Fisher C. Beglova N. Blacklow S.C. Mol. Cell. 2006; 22: 277-283Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar and by Lee et al., 2006Lee D. Walsh J.D. Mikhailenko I. Yu P. Migliorini M. Wu Y. Krueger S. Curtis J.E. Harris B. Lockett S. et al.Mol. Cell. 2006; 22: 423-430Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar have now opened the door to a deeper and comprehensive understanding of the mechanisms by which a specialized chaperone controls the expression of an ancient and functionally highly diverse family of cell surface receptors. Structure of an LDLR-RAP Complex Reveals a General Mode for Ligand Recognition by Lipoprotein ReceptorsFisher et al.Molecular CellApril 21, 2006In BriefProteins of the low-density lipoprotein receptor (LDLR) family are remarkable in their ability to bind an extremely diverse range of protein and lipoprotein ligands, yet the basis for ligand recognition is poorly understood. Here, we report the 1.26 Å X-ray structure of a complex between a two-module region of the ligand binding domain of the LDLR and the third domain of RAP, an escort protein for LDLR family members. The RAP domain forms a three-helix bundle with two docking sites, one for each LDLR module. Full-Text PDF Open ArchiveHistone H3 and H4 Ubiquitylation by the CUL4-DDB-ROC1 Ubiquitin Ligase Facilitates Cellular Response to DNA DamageWang et al.Molecular CellMay 05, 2006In BriefPosttranslational histone modifications play important roles in transcription and other chromatin-based processes. Compared to acetylation, methylation, and phosphorylation, very little is known about the function of histone ubiquitylation. Here, we report the purification and functional characterization of a histone H3 and H4 ubiquitin ligase complex, CUL4-DDB-ROC1. We demonstrate that CUL4-DDB-ROC1-mediated H3 and H4 ubiquitylation occurs both in vitro and in vivo. Importantly, CUL4-DDB-ROC1-mediated H3 and H4 ubiquitylation is regulated by UV irradiation. Full-Text PDF Open Archive