CD91, also known as the low density lipoprotein receptor-related protein 1 (LRP1), is a receptor on the plasma membrane involved in receptor-mediated endocytosis and in various signaling events. CD91 is a member of a gene family found in diverse species, including C. elegans, Drosophila, Xenopus, and mammals (Gaultier et al. 2008). Seven structurally closely related cell surface receptors constitute the core of the low density lipoprotein (LDL) receptor gene family. They include the LDL receptor, the LDL receptor-related protein (CD91), LRP1b, megalin, very low density lipoprotein (VLDL) receptor, apolipoprotein E receptor 2 (apo-ER2), and multiple epidermal growth factor-like domains containing protein 7 (MEGF7) (Herz and Strickland 2001). This review focuses on CD91 and its role as a signaling receptor for several ligands and in various physiological processes.
CD91 was originally identified by virtue of its structural similarity and sequence homology to the LDL receptor (Herz et al. 1988). As expected of a lipoprotein receptor, CD91 was found to play an important role in lipoprotein metabolism and cholesterol homeostasis by the discovery of apolipoprotein E as its ligand. The function of CD91 in vivo as a chylomicron remnant receptor is now well established. The sequencing of the receptor soon revealed that it was identical to the receptor proposed for removal of α2M-proteinase complexes (Lillis et al. 2008).
CD91 is synthesized as a 600-kDa type I transmembrane protein which during transit to the cell surface is processed by furin within a β-propeller domain into a large 515-kDa α- and a smaller 85-kDa β -subunit in the trans-Golgi network. An ER-resident chaperone, termed the receptor-associated protein (RAP), binds with high affinity to CD91 on multiple sites and prevents premature association of ligands to the newly synthesized CD91 in the ER enabling it to be successfully delivered to the plasma membrane (Lillis et al. 2008).
CD91 is composed of modular structures that include cysteine-rich complement-type repeats, EGF repeats, β-propeller domains, all on the α-chain, and a transmembrane and cytoplasmic domain on the β-chain. The cysteine-rich complement-type repeats, also commonly referred to as ligand-binding repeats, are localized into regions as clusters and are termed clusters I–IV, each containing variable numbers of complement-type repeats. Most CD91 ligands have been shown to bind to clusters II and IV (Lillis et al. 2008).
The β-chain contains a small extracellular domain, a single-pass transmembrane domain, and an intracellular domain of 100 amino acid residues. After furin cleavage in the ER, the β-chain stays noncovalently connected to the larger α-subunit through its small extracellular domain. The intracellular domain of CD91 contains many motifs which are postulated to have a role in basolateral sorting, internalization, recycling of the receptor, and binding of several different adaptor proteins for signal transduction. These motifs on the β-chain include two NPXY motifs, the distal one overlapping with an YXXL internalization motif, two di-leucine internalization motifs, and several serine, threonine, and tyrosine phosphorylation sites. Unlike the other LDL receptors, the YXXL motif rather than the NPXY and di-leucine motifs was shown to be the dominant endocytosis signal in CD91 mini-receptor constructs (Li et al. 2000).
The receptor is expressed in a variety of cell types, including macrophages (Misra et al. 1995), dendritic cells (Basu et al. 2001; Messmer et al. 2013), T helper cells (Banerjee et al. 2002), adrenal cortical cells, hepatocytes, neurons and neuroblastoma, follicular cells of the ovary, fibroblasts, mesangial cells (Zheng et al. 1994), and adipocytes (Corvera et al. 1989).
The Role of CD91 in Cell Signaling
A significant number of biological functions have been attributed to CD91 due to its ability to bind to a large number of ligands. Mice deficient in CD91 expression die early during embryonic development demonstrating a critical function for CD91 in prenatal development (Herz and Strickland 2001). Mice engineered to lack expression of CD91 in vascular smooth muscle cells display overexpression of, and abnormal signaling through, the platelet-derived growth factor (PDGF) receptor which causes proliferation of smooth muscle cells, aneurysm formation, and marked susceptibility to cholesterol-induced atherosclerosis (Boucher et al. 2003). Generally the functions of CD91 are mediated through two major physiological processes, endocytosis and initiation and/or regulation of signaling pathways. During endocytosis, CD91 is capable of binding approximately 30 ligands with high affinity and delivering them to endo-lysosomal compartments. The receptor itself is usually recycled. The details of CD91-mediated endocytosis are outside the scope of this review.
The role of CD91 signaling in apoptosis: CD91 is one of the numerous cell surface receptors that have been shown to engage apoptotic cells and promote phagocytosis (Kinchen and Ravichandran 2007). CD91 was shown to be a receptor for calreticulin, a Ca2+-binding protein normally found in the endoplasmic reticulum, in 2001 (Basu et al. 2001). Those experiments were performed with soluble calreticulin in the extracellular space. In an extension of those studies, CD91 has been shown to bind calreticulin on the surface of apoptotic cells which in turn directly interacts with phosphatidylserine on the apoptotic cell surface. These interactions are important for clearance of apoptotic cells by signaling an “eat me” signal within phagocytic cells such as macrophages (Gardai et al. 2006). The nature of the signaling pathway with respect to CD91 and apoptosis is currently unknown. Interestingly, CD91 also serves as an antiapoptotic receptor in neurons and macrophages via activation of PI3K/Akt and GSK-3β (Fuentealba et al. 2009; Yancey et al. 2010). The role of the PI3K/Akt signaling pathway in cell survival has been well documented and is required for proliferation and growth factor activity (Dellinger and Brekken 2011). Fuentealba et al. found that deletion of CD91 in mouse primary neurons results in decreased survival of cells upon treatment of neurotoxic agent Aβ42 due to loss of Akt phosphorylation (Fuentealba et al. 2009). Similarly, CD91-deficient macrophages exposed to apoptotic conditions have decreased Akt phosphorylation and increased expression of pro-inflammatory cytokines IL-1β and IL-6 (Yancey et al. 2010).
The role of CD91 signaling in immune cells: The HSPs, gp96 (Grp94), hsp70, hsp90, and calreticulin are immunogenic. Upon immunization with HSPs, frogs, mice, rats, and humans elicit immune responses to peptides that are chaperoned by the HSP (Binder 2008). Upon examination of the mechanism of action, CD91 was first identified by Binder et al. as the receptor for gp96 (Binder et al. 2000) and then by Basu et al. as a common receptor for other immunogenic HSPs such as hsp70, hsp90, and calreticulin (Basu et al. 2001). Utilizing antigen-presenting cells in the form of dendritic cells or macrophages, CD91 has been demonstrated in numerous laboratories to be essential for endocytosis of HSP-peptide complexes and cross-presentation of the chaperoned peptide. While the role of CD91-dependent endocytosis in HSP-mediated immunity is well established, its role in signaling has also been examined (Pawaria and Binder 2011). It appears that in response to binding any one of the immunogenic HSPs, gp96, hsp70, or calreticulin, (i) CD91 is phosphorylated; (ii) CD91 associates with the Shc adaptor protein, a process that involves the Src family kinases; and (iii) p38 MAP kinase and NF-κB is activated in a CD91-dependent manner. This signaling pathway is important for the maturation of antigen-presenting cells and the resulting elaboration of costimulation for T cell priming. Dendritic cells in which CD91 is deleted do not respond to gp96. This is consistent with a lack of immune responses in conditional knock-out mice in which CD91 is eliminated in CD11c+ cells and immunized with gp96 (Zhou et al. 2014). Current studies examining other kinases are the focus of several laboratories. For example, p38 MAP kinases which are activated in response to HSP-CD91 interaction appear to act as upstream mediators of STAT1 signaling (Kinner-Bibeau and Binder, personal communication). Peritoneal macrophages produce several STAT1-dependent cytokines in response to gp96, including IP10 and RANTES (Sedlacek et al. 2016). CD91 thus appears to play a dual role in HSP-mediated immunity, provision of signal 1 by way of cross-presentation of antigens and signal 2 via costimulation (Fig. 1). These events play key roles in initiation of antitumor immune responses in tumor-bearing mice (Zhou et al. 2014) and for therapeutic purposes in the clinic (Srivastava 2006).
CD91 as a transcriptional regulator: It has been recognized for some time that a soluble form of CD91 circulates in the plasma, indicating that the receptor is subject to proteolysis, thereby releasing portions of receptor as a soluble polypeptide (Quinn et al. 1999). Similar to other type I integral membrane proteins, the CD91 cytoplasmic domain can also be cleaved by an enzyme with γ-secretase-like activity (May et al. 2002) releasing CD91 polypeptides of about 12 kDa into the cytosol. This polypeptide has been shown to translocate to the nucleus where it limits the transcription of the inflammatory genes TNF-α and IL-6 in response to LPS activation of cells (Zurhove et al. 2008). However, a nuclear localization signal within this polypeptide is yet to be defined. Briefly, the nuclear CD91 polypeptide colocalizes with histone acetyltransferase Tip60, a transcriptional modulator with a role in linking proteolytic cleavage of β-amyloid precursor protein (APP) to transcriptional activation (Cao and Sudhof 2001; Baek et al. 2002). These studies suggest that a portion of the β-chain of CD91 can function directly as a transcriptional regulator. The identification of other genes regulated in a similar mechanism by this polypeptide is expected.
The role of CD91 signaling in the nervous system: Bu et al. found that the cytoplasmic tail of CD91 is phosphorylated in neuronal-derived cell lines and that nerve growth factor rapidly increases the amount of phosphorylation (Bu et al. 1998). A result of this is the rapid increase of cell surface CD91. CD91 serves as a receptor for α2M with or without the amyloid peptide apolipoprotein E, tissue factor pathway inhibitor, and APP among others. These proteins are associated with the onset or pathology of Alzheimer’s disease. An increase in CD91 expression on neurons suggests a mechanism for increased clearance of these proteins in the brain. In addition, signaling through phosphorylation of CD91 has been suggested to be crucial for the redistribution of CD91 from a silent pool (in the endosomes) to an active endocytic pool (on the cell surface). Redistribution of CD91 in neurons by this mechanism serves as a rapid way to regulate concentration of its ligands in the local environment of the brain. CD91 also promotes axonal regeneration and neurite outgrowth via activation of TrkC, Akt, and ERK (Yoon et al. 2013).
The role of CD91 in metabolism: Uptake, processing, and accumulation of cholesterol is highly regulated by CD91 in multiple cell types including vascular smooth muscle cells and macrophages (Llorente-Cortés et al. 2007; Lillis et al. 2015). CD91-dependent cholesterol storage and fatty acid metabolism are regulated through canonical Wnt5a- and p-GSK3β-signaling pathways (Terrand et al. 2009). When CD91 expression is abrogated in fibroblasts, cholesterol storage is increased at least in part due to impaired Wnt signaling. Lack of Wnt5a signaling in these cells also enhanced phosphorylation of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme for fatty acid synthesis. This event occurs through a GSK-3β-dependent pathway that does not involve p-AMPK. CD91 is also implicated in glucose metabolism and insulin signaling, via regulation of insulin receptor β expression and control of p-Akt levels in response to insulin (Liu et al. 2015).
Other signaling events that have not been associated with a physiological outcome: These studies have largely been performed on cells solely in an in vitro setting, and thus their role in physiological effects is not yet readily apparent. Barnes et al. identified the CD91 β-chain bound to Shc PTB domain and showed that CD91 is tyrosine-phosphorylated in v-Src transformed cells. The tyrosine-phosphorylated CD91 binds to Shc which might provide a link to the RAS-ERK/MAP kinase pathway and alternative pathways downstream of Shc (Barnes et al. 2001). Those results are consistent with our own observations on the role of CD91 in immune cells described above. Tyrosine phosphorylation of CD91 suggested that a member of the receptor tyrosine kinase family might be responsible for this effect. Loukinova et al. found that PDGFBB induces a transient tyrosine phosphorylation of the CD91 cytoplasmic domain in a process dependent on PDGFβ receptor activation and c-Src family kinase activity (Loukinova et al. 2002). Lastly, CD91 is phosphorylated on cytoplasmic tyrosines in response to platelet-derived growth factor (Boucher et al. 2002), and Tyr 4507 was identified as the principle v-Src phosphorylation site (Barnes et al. 2001).
The endocytic functions of CD91 have been avidly examined and described. With respect to over 30 ligands described to bind to the α-chain of CD91, most have been demonstrated to be endocytosed into endo-lysosomes. There are an increasing number of examples of important roles for signaling via CD91 in various physiological events as described here. While an association of CD91 and its ligands in Alzheimer’s disease has been studied for a number of years, the role of CD91 in immune responses is only beginning to emerge. In other physiological events, the crucial role of CD91 in clearing apoptotic cells signifies the far reaching consequences a deficiency of CD91 function would have. Indeed no such deficiencies have been found. In our own laboratory, signaling of CD91 in response to immunogenic HSPs is a critical pathway for initiation of immune responses by dendritic cells and also for directing the type of immune response that is primed. This ancient receptor that has a major role in endocytosis appears to have been hijacked for evolutionarily newer functions of higher organisms. A major challenge for the study of CD91 is its sheer size of 600 kDa and the intricate conformation of its domains which makes expression of recombinant protein with the right protein conformation challenging. In addition, mice that are created to be deficient in CD91 expression die early during embryonic development due to its role in prenatal development (Herz and Strickland 2001). Recent advances in artificial chromosomes and conditional knock-out mice technology using the Cre-lox system (Zhou et al. 2014) have provided invaluable reagents for further investigation into the role in pathology and pathogenesis of disease and the role of CD91 signaling in physiological pathways.
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