Ligand-specific Conformational Changes in the ��1 Glycine Receptor Ligand-binding Domain

Understanding the activation mechanism of Cys loop ion channel receptors is key to understanding their physiological and pharmacological properties under normal and pathological conditions. The ligand-binding domains of these receptors comprise inner and outer β-sheets and structural studies indicate that channel opening is accompanied by conformational rearrangements in both β-sheets. In an attempt to resolve ligand-dependent movements in the ligand-binding domain, we employed voltage-clamp fluorometry on α1 glycine receptors to compare changes mediated by the agonist, glycine, and by the antagonist, strychnine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. In the inner β-sheet, we labeled residues in loop 2 and in binding domain loops D and E. At each position, strychnine and glycine induced distinct maximal fluorescence responses. The pre-M1 domain responded similarly; at each of four labeled positions glycine produced a strong fluorescence signal, whereas strychnine did not. This suggests that glycine induces conformational changes in the inner β-sheet and pre-M1 domain that may be important for activation, desensitization, or both. In contrast, most labeled residues in loops C and F yielded fluorescence changes identical in magnitude for glycine and strychnine. A notable exception was H201C in loop C. This labeled residue responded differently to glycine and strychnine, thus underlining the importance of loop C in ligand discrimination. These results provide an important step toward mapping the domains crucial for ligand discrimination in the ligand-binding domain of glycine receptors and possibly other Cys loop receptors.Glycine receptor (GlyR)3 chloride channels are pentameric Cys loop receptors that mediate fast synaptic transmission in the nervous system (1, 2). This family also includes nicotinic acetylcholine receptors (nAChRs), γ-aminobutyric acid type A and type C receptors, and serotonin type 3 receptors. Individual subunits comprise a large ligand-binding domain (LBD) and a transmembrane domain consisting of four α-helices (M1–M4). The LBD consists of a 10-strand β-sandwich made of an inner β-sheet with six strands and an outer β-sheet with four strands (3). The ligand-binding site is situated at the interface of adjacent subunits and is formed by loops A–C from one subunit and loops D–F from the neighboring subunit (3).The activation mechanism of Cys loop receptors is currently the subject of intense investigation because it is key to understanding receptor function under normal and pathological conditions (4, 5). Based on structural analysis of Torpedo nAChRs, Unwin and colleagues (6, 7) originally proposed that agonist binding induced the inner β-sheet to rotate, whereas the outer β-sheet tilted slightly upwards with loop C clasping around the agonist. These movements were thought to be transmitted to the transmembrane domain via a differential movement of loop 2 (β1-β2) and loop 7 (β6-β7) (both part of the inner β-sheet) and the pre-M1 domain (which is linked via a β-strand to the loop C in the outer sheet). The idea of large loop C movements accompanying agonist binding is supported by structural and functional data (3, 8–13). However, a direct link between loop C movements and channel gating has proved more difficult to establish. Although computational modeling studies have suggested that this loop may be a major component of the channel opening mechanism (14–18), experimental support for this model is not definitive. Similarly, loop F is also thought to move upon ligand binding, although there is as yet no consensus as to whether these changes represent local or global conformational changes (11, 19–21). Recently, a comparison of crystal structures of bacterial Cys loop receptors in the closed and open states revealed that although both the inner and outer β-sheets exhibit different conformations in closed and open states, the pre-M1 domain remains virtually stationary (22, 23). It is therefore relevant to question whether loop C, loop F, and pre-M1 movements are essential for Cys loop receptor activation.Strychnine is a classical competitive antagonist of GlyRs (24, 25), and to date there is no evidence that it can produce LBD structural changes. In this study we use voltage-clamp fluorometry (VCF) to compare glycine- and strychnine-induced conformational changes in the GlyR loops 2, C, D, E, and F and the pre-M1 domain in an attempt to determine whether they signal ligand-binding events, local conformational changes, or conformational changes associated with receptor activation.In a typical VCF experiment, a domain of interest is labeled with an environmentally sensitive fluorophore, and current and fluorescence are monitored simultaneously during ligand application. VCF is ideally suited for identifying ligand-specific conformational changes because it can report on electrophysiologically silent conformational changes (26), such as those induced by antagonists. Indeed, VCF has recently provided valuable insights into the conformational rearrangements of various Cys loop receptors (19, 21, 27–33).     

The Linker Region in Receptor Guanylyl Cyclases Is a Key Regulatory Module: MUTATIONAL ANALYSIS OF GUANYLYL CYCLASE C*

Receptor guanylyl cyclases are multidomain proteins, and ligand binding to the extracellular domain increases the levels of intracellular cGMP. The intracellular domain of these receptors is composed of a kinase homology domain (KHD), a linker of ∼70 amino acids, followed by the C-terminal guanylyl cyclase domain. Mechanisms by which these receptors are allosterically regulated by ligand binding to the extracellular domain and ATP binding to the KHD are not completely understood. Here we examine the role of the linker region in receptor guanylyl cyclases by a series of point mutations in receptor guanylyl cyclase C. The linker region is predicted to adopt a coiled coil structure and aid in dimerization, but we find that the effects of mutations neither follow a pattern predicted for a coiled coil peptide nor abrogate dimerization. Importantly, this region is critical for repressing the guanylyl cyclase activity of the receptor in the absence of ligand and permitting ligand-mediated activation of the cyclase domain. Mutant receptors with high basal guanylyl cyclase activity show no further activation in the presence of non-ionic detergents, suggesting that hydrophobic interactions in the basal and inactive conformation of the guanylyl cyclase domain are disrupted by mutation. Equivalent mutations in the linker region of guanylyl cyclase A also elevated the basal activity and abolished ligand- and detergent-mediated activation. We, therefore, have defined a key regulatory role for the linker region of receptor guanylyl cyclases which serves as a transducer of information from the extracellular domain via the KHD to the catalytic domain.In transmembrane receptors a series of conformational changes are required to transmit the information of ligand binding (an extracellular signal) to the interior of the cell, resulting in either altered interaction with signaling intermediates or in the regulation of a catalytic activity present in the receptor. In these multidomain receptors, where the ligand binding and effector domains are present in the same polypeptide chain, the relay of conformational changes is under the exquisite control of post-translational modifications or precise structural alterations.Receptor guanylyl cyclases (GCs)⁴ have an N-terminal extracellular ligand binding domain, a single transmembrane domain, and a C-terminal intracellular domain (1). Binding of ligands to the extracellular domain elicits a conformational change that increases the guanylyl cyclase activity of the receptor, resulting in increased cGMP production. The intracellular domain of receptor GCs contains a region that shares considerable sequence similarity to protein kinases and is referred to as the kinase homology domain (KHD). Binding of ATP to the KHD induces a conformational change that regulates cGMP production by the guanylyl cyclase domain (2). Thus, receptor GCs exemplify the intricate interactions between domains in transducing the signal from an extracellular ligand to the interior of the cell.The amino acid sequences of the extracellular domain of mammalian receptor GCs vary (less than ∼15% similarity), as would be expected given the diversity in the ligands that bind to and activate these receptors. The KHD shows ∼25–30% conservation in amino acid sequence across receptor GCs, and computational modeling has not only suggested that this region could adopt the overall structure of a protein kinase but also identified specific residues that could interact with ATP (2, 3). The catalytic domains of mammalian receptor GCs are more conserved (∼80% sequence similarity). The gradual increase in sequence similarity across the various domains, with the extracellular domain being the most diverse and the cyclase domains sharing the maximum sequence similarity, is a reflection of the ability of these receptor GCs to converge diverse extracellular signals to a unified output of cGMP production. The guanylyl cyclase domains of receptor GCs can be classified as members of the Class III family of nucleotide cyclases (4). The recent crystal structures of a bacterial guanylyl cyclase (5) and a eukaryotic soluble guanylyl cyclase (6) show similarities in the overall three-dimensional structure of adenylyl and guanylyl cyclases and also highlight the critical residues that determine substrate utilization (either ATP or GTP) in these enzymes.Guanylyl cyclase C (GC-C) serves as the receptor for the guanylin family of endogenous peptides as well as for the exogenous heat-stable enterotoxin (ST) peptides secreted by enterotoxigenic bacteria (7, 8). GC-C is predominantly expressed on the apical surface of epithelial cells in the intestine, although robust extra-intestinal expression is observed in the kidney and reproductive tissues of the rat (9–12). The extracellular domain of GC-C is glycosylated, and we have shown the importance of glycosylation in regulating receptor desensitization in colonic cells. We have also identified a critical residue (Lys-516) in the KHD of GC-C as being important for KHD-mediated modulation of the guanylyl cyclase activity (2, 3).A sequence of ∼70 amino acids is found between the KHD and the guanylyl cyclase domain of receptor GCs, which we refer to here as the linker region (13). This region is predicted to form an amphipathic α-helix and could also adopt a coiled coil conformation (14, 15). The linker region is also present in soluble (cytosolic) guanylyl cyclases where it connects the N-terminal heme binding regulatory domain to the C-terminal catalytic cyclase domain. The linker region is suggested to act as a dimerization module in receptor GCs (16–18) and has also been implicated in heterodimerization of the α and β subunits of soluble guanylyl cyclases (19, 20). However, there are several reports to the contrary that indicate that the linker does not affect the dimerization of receptor GCs (14, 15). Nevertheless, the critical importance of the linker in regulating the activity of receptor GCs is shown by the fact that mutations in this region of the retinal guanylyl cyclase (RetGC-1) are associated with autosomal dominant cone-rod dystrophy in humans (16, 21). We show here through extensive mutational and biochemical analysis that the linker regions in two receptor GCs, GC-C and guanylyl cyclase A (GC-A), play an important role in repressing the catalytic activity of the receptors in the absence of their ligands. In addition, our results provide for the first time a molecular explanation for detergent-enhanced guanylyl cyclase activity in this family of receptors and suggest a mechanism for this activation that could involve a hydrophobic interaction between the linker region and the guanylyl cyclase domain.     

Ubiquitination Regulates Proteolytic Processing of G Protein-coupled Receptors after Their Sorting to Lysosomes

Ubiquitination is essential for the endocytic sorting of various G protein-coupled receptors to lysosomes. Here we identify a distinct function of this covalent modification in controlling the later proteolytic processing of receptors. Mutation of all cytoplasmic lysine residues in the murine δ-opioid receptor blocked receptor ubiquitination without preventing ligand-induced endocytosis of receptors or their subsequent delivery to lysosomes, as verified by proteolysis of extramembrane epitope tags and down-regulation of radioligand binding to the transmembrane helices. Surprisingly, a functional screen revealed that the E3 ubiquitin ligase AIP4 specifically controls down-regulation of wild type receptors measured by radioligand binding without detectably affecting receptor delivery to lysosomes defined both immunochemically and biochemically. This specific AIP4-dependent regulation required direct ubiquitination of receptors and was also regulated by two deubiquitinating enzymes, AMSH and UBPY, which localized to late endosome/lysosome membranes containing internalized δ-opioid receptor. These results identify a distinct function of AIP4-dependent ubiquitination in controlling the later proteolytic processing of G protein-coupled receptors, without detectably affecting their endocytic sorting to lysosomes. We propose that ubiquitination or ubiquitination/deubiquitination cycling specifically regulates later proteolytic processing events required for destruction of the receptor''s hydrophobic core.A fundamental cellular mechanism contributing to homeostatic regulation of receptor-mediated signal transduction involves ligand-induced endocytosis of receptors followed by proteolysis in lysosomes. The importance of such proteolytic down-regulation has been documented extensively for a number of seven-transmembrane or G protein-coupled receptors (GPCRs),³ which comprise the largest known family of signaling receptors expressed in animals, as well as for other important signaling receptors, such as the epidermal growth factor receptor tyrosine kinase (1–5).One GPCR that is well known to undergo endocytic trafficking to lysosomes is the δ-opioid peptide receptor (DOR or DOP-R) (6). Following endocytosis, DOR traffics efficiently to lysosomes in both neural and heterologous cell models (6–8), whereas many membrane proteins, including various GPCRs, recycle rapidly to the plasma membrane (9–12). Such molecular sorting of internalized receptors between divergent recycling and degradative pathways is thought to play a fundamental role in determining the functional consequences of regulated endocytosis (2, 3, 13, 14). The sorting process that directs internalized DOR to lysosomes is remarkably efficient and appears to occur rapidly (within several min) after receptor endocytosis (11). Nevertheless, biochemical mechanisms that control lysosomal trafficking and proteolysis of DOR remain poorly understood.A conserved mechanism that promotes lysosomal trafficking of a number of membrane proteins, including various signaling receptors, is mediated by covalent modification of cytoplasmic lysine residues with ubiquitin (4, 15–17). Ubiquitination was first identified as an endocytic sorting determinant in studies of vacuolar trafficking of the yeast GPCR Ste2p (18). Subsequent studies have established numerous examples of lysyl-ubiquitination being required for sorting endocytic cargo to lysosomes and have identified conserved machinery responsible for the targeting of ubiquitinated cargo to lysosomes (3, 17, 19–22).The CXCR4 chemokine receptor provides a clear example of ubiquitin-dependent lysosomal sorting of a mammalian GPCR. Ubiquitination of the carboxyl-terminal cytoplasmic domain of the CXCR4 receptor, mediated by the E3 ubiquitin ligase AIP4, is specifically required for the HRS- and VPS4-dependent trafficking of internalized receptors to lysosomes. Blocking this ubiquitination event by Lys → Arg mutation of the receptor specifically inhibits trafficking of internalized receptors to lysosomes, resulting in recycling rather than lysosomal proteolysis of receptors after ligand-induced endocytosis (23–25).Lysosomal trafficking of DOR, in contrast, is not prevented by mutation of cytoplasmic lysine residues (26) and can be regulated by ubiquitination-independent protein interaction(s) (27, 28). Nevertheless, both wild type and lysyl-mutant DORs traffic to lysosomes via a similar pathway as ubiquitin-dependent membrane cargo and require both HRS and active VPS4 to do so (29). These observations indicate that DOR engages the same core endocytic mechanism utilized by ubiquitination-directed membrane cargo but leave unresolved whether ubiquitination of DOR plays any role in this important cellular mechanism of receptor down-regulation.There is no doubt that DOR can undergo significant ubiquitination in mammalian cells, including HEK293 cells (30–32), where lysosomal trafficking of lysyl-mutant receptors was first observed (26). Ubiquitination was shown previously to promote proteolysis of DOR by proteasomes and to function in degrading misfolded receptors from the biosynthetic pathway (30, 31). A specific role of ubiquitination in promoting proteasome- but not lysosome-mediated proteolysis of DOR has been emphasized (32) and proposed to contribute to proteolytic down-regulation of receptors also from the plasma membrane (33).To our knowledge, no previous studies have determined if DOR ubiquitination plays any role in controlling receptor proteolysis mediated by lysosomes, although this represents a predominant pathway by which receptors undergo rapid down-regulation following ligand-induced endocytosis in a number of cell types, including HEK293 cells (8). In the present study, we have taken two approaches to addressing this fundamental question. First, we have investigated in greater detail the effects of lysyl-mutation on DOR ubiquitination and trafficking. Second, we have independently investigated the role of ubiquitination in controlling lysosomal proteolysis of wild type DOR. Our results clearly establish the ability of DOR to traffic efficiently to lysosomes in the absence of any detectable ubiquitination. Further, they identify a distinct and unanticipated function of AIP4-dependent ubiquitination in regulating the later proteolytic processing of receptors and show that this distinct ubiquitin-dependent regulatory mechanism operates effectively downstream of the sorting decision that commits internalized receptors for delivery to lysosomes.     

Quantitative Proteome Analysis of Temporally Resolved Phagosomes Following Uptake Via Key Phagocytic Receptors

Macrophages operate at the forefront of innate immunity and their discrimination of foreign versus “self” particles is critical for a number of responses including efficient pathogen killing, antigen presentation, and cytokine induction. In order to efficiently destroy the particles and detect potential threats, macrophages express an array of receptors to sense and phagocytose prey particles. In this study, we accurately quantified a proteomic time-course of isolated phagosomes from murine bone marrow-derived macrophages induced by particles conjugated to seven different ligands representing pathogen-associated molecular patterns, immune opsonins or apoptotic cell markers. We identified a clear functional differentiation over the three timepoints and detected subtle differences between certain ligand-phagosomes, indicating that triggering of receptors through a single ligand type has mild, but distinct, effects on phagosome proteome and function. Moreover, our data shows that uptake of phosphatidylserine-coated beads induces an active repression of NF-κB immune responses upon Toll-like receptor (TLR)-activation by recruitment of anti-inflammatory regulators to the phagosome. This data shows for the first time a systematic time-course analysis of bone marrow-derived macrophages phagosomes and how phagosome fate is regulated by the receptors triggered for phagocytosis.Macrophages exist in many different tissue subsets, are extremely plastic in response to cytokines and pathogen-associated molecular patterns and perform a wide range of biological functions (1, 2). One of the most important functions of macrophages is phagocytosis, defined as the active uptake of large particles (>0.5 μm) by cells (3). Phagocytosis is an important cellular mechanism for almost all eukaryotes, highly conserved in evolution (4), and, in mammals, is a key part of the innate immune response to invading microorganisms. Moreover, during homeostasis and development, macrophages phagocytose apoptotic cells and cell debris to recycle cellular building blocks (5, 6). Phagocytosis is induced through the binding of particles as diverse as microbes, apoptotic cells, or even inert beads to cell surface receptors. After internalization, newly formed phagosomes engage in a maturation process that involves fusion with various organelles, including endosomes and ultimately lysosomes (7, 8). This leads to the formation of phagolysosomes that degrade the foreign matter. Antigens from the particle are presented via MHC class I and II molecules, bridging innate and adaptive immunity.In order to effectively phagocytose the diverse types of particulates they can encounter, macrophages express a vast array of receptors to sense and respond to the different ligands; however, only a small subset are solely sufficient to trigger phagocytosis (9). The classic phagocytic receptors are the Fc receptor, which internalizes immunoglobulin-bound particles (10), and the complement receptors, which binds to complement-opsonized particles (11). Other well characterized ligands for phagocytic receptors include mannan, a polysaccharide common in bacterial membranes and fungal cell walls (12), that activates mannose receptors (13, 14); lipopolysaccharide (LPS)¹, a glycolipid that constitutes the major portion of the outermost membrane of Gram-negative bacteria, that triggers CD14 as well as scavenger receptors and toll-like receptors (15–20); and phosphatidylserine (PS), a lipid normally restricted to the inner leaflet of eukaryotic plasma membranes, but exposed in the outer leaflet during apoptosis. PS provides an “eat me” signal for macrophage clearance (21, 22) and triggers a range of receptors including TIM-4, BAI1, and Stabilin-2 (23–27). Similarly, calreticulin, an endoplasmic reticulum protein that is also transported to the plasma membrane serves as a apoptotic signal has been proposed as a phagocytic ligand triggering the phagocytic receptor low-density lipoprotein receptor-related protein (LRP) (28–30).Although it is established that phagosome function is affected by various activation states, including rate of maturation, degradative capacity, and antigen cross-presentation capabilities (31–33), controversy exists around whether phagosome activity can be controlled directly, without prior activation, by receptor engagement at the phagosome level during biogenesis (34–38). Here, we dissect the role that individual ligands play in controlling downstream phagosome maturation using a reductionist strategy of ligating single ligands to microparticles and analyzing resulting phagosomes by quantitative proteomics and fluorescent phagosome functional assays.     

Purification and Functional Reconstitution of Monomeric ��-Opioid Receptors: ALLOSTERIC MODULATION OF AGONIST BINDING BY Gi2*

Despite extensive characterization of the μ-opioid receptor (MOR), the biochemical properties of the isolated receptor remain unclear. In light of recent reports, we proposed that the monomeric form of MOR can activate G proteins and be subject to allosteric regulation. A μ-opioid receptor fused to yellow fluorescent protein (YMOR) was constructed and expressed in insect cells. YMOR binds ligands with high affinity, displays agonist-stimulated [³⁵S]guanosine 5′-(γ-thio)triphosphate binding to Gα_i, and is allosterically regulated by coupled G_i protein heterotrimer both in insect cell membranes and as purified protein reconstituted into a phospholipid bilayer in the form of high density lipoprotein particles. Single-particle imaging of fluorescently labeled receptor indicates that the reconstituted YMOR is monomeric. Moreover, single-molecule imaging of a Cy3-labeled agonist, [Lys⁷, Cys⁸]dermorphin, illustrates a novel method for studying G protein-coupled receptor-ligand binding and suggests that one molecule of agonist binds per monomeric YMOR. Together these data support the notion that oligomerization of the μ-opioid receptor is not required for agonist and antagonist binding and that the monomeric receptor is the minimal functional unit in regard to G protein activation and strong allosteric regulation of agonist binding by G proteins.Opioid receptors are members of the G protein-coupled receptor (GPCR)² superfamily and are clinical mainstays for inducing analgesia. Three isoforms of opioid receptors, μ, δ, and κ, have been cloned and are known to couple to G_i/o proteins to regulate adenylyl cyclase and K⁺/Ca⁺ ion channels (1–3). An ever growing amount of data suggests that many GPCRs oligomerize (4, 5), and several studies have suggested that μ-opioid receptors (MORs) and δ-opioid receptors heterodimerize to form unique ligand binding and G protein-activating units (6–10). Although intriguing, these studies utilize cellular overexpression systems where it is difficult to know the exact nature of protein complexes formed between the receptors.To study the function of isolated GPCRs, our laboratory and others have utilized a novel phospholipid bilayer reconstitution method (11–16). In this approach purified GPCRs are reconstituted into the phospholipid bilayer of a high density lipoprotein (HDL) particle. The reconstituted HDL (rHDL) particles are monodispersed, uniform in size, and preferentially incorporate a GPCR monomer (14, 15). Previous work in our lab has shown that rhodopsin, a class A GPCR previously proposed to function as a dimer (17–19), is fully capable of activating its G protein when reconstituted as a monomer in the rHDL lipid bilayer (15). Moreover, we have demonstrated that agonist binding to a monomeric β₂-adrenergic receptor, another class A GPCR, can be allosterically regulated by G proteins (14). This led us to determine whether a monomer of MOR, a class A GPCR that endogenously binds peptide ligands, is the minimal functional unit required to activate coupled G proteins. We additionally investigated whether agonist binding to monomeric MOR is allosterically regulated by inhibitory G protein heterotrimer.To study the function of monomeric MOR we have purified a modified version of the receptor to near homogeneity. A yellow fluorescent protein was fused to the N terminus of MOR, and this construct (YMOR) was expressed in insect cells for purification. After reconstitution of purified YMOR into rHDL particles, single-molecule imaging of Cy3-labeled and Cy5-labeled YMOR determined that the rHDL particles contained one receptor. This monomeric YMOR sample binds ligands with affinities nearly equivalent to those observed in plasma membrane preparations. Monomeric YMOR efficiently stimulates GTPγS binding to G_i2 heterotrimeric G protein. G_i2 allosteric regulation of agonist binding to rHDL·YMOR was also observed. Single-particle imaging of binding of [Lys⁷, Cys⁸]dermorphin-Cy3, a fluorophore-labeled agonist, to rHDL·YMOR supports the notion that the rHDL particles contain a single YMOR. Taken together, these results suggest that a monomeric MOR is the minimal functional unit for ligand binding and G protein activation and illustrate a novel method for imaging ligand binding to opioid receptors.     

Infection of Glial Cells by the Human Polyomavirus JC Is Mediated by an N-Linked Glycoprotein Containing Terminal α(2-6)-Linked Sialic Acids

The human JC polyomavirus (JCV) is the etiologic agent of the fatal central nervous system (CNS) demyelinating disease progressive multifocal leukoencephalopathy (PML). PML typically occurs in immunosuppressed patients and is the direct result of JCV infection of oligodendrocytes. The initial event in infection of cells by JCV is attachment of the virus to receptors present on the surface of a susceptible cell. Our laboratory has been studying this critical event in the life cycle of JCV, and we have found that JCV binds to a limited number of cell surface receptors on human glial cells that are not shared by the related polyomavirus simian virus 40 (C. K. Liu, A. P. Hope, and W. J. Atwood, J. Neurovirol. 4:49–58, 1998). To further characterize specific JCV receptors on human glial cells, we tested specific neuraminidases, proteases, and phospholipases for the ability to inhibit JCV binding to and infection of glial cells. Several of the enzymes tested were capable of inhibiting virus binding to cells, but only neuraminidase was capable of inhibiting infection. The ability of neuraminidase to inhibit infection correlated with its ability to remove both α(2-3)- and α(2-6)-linked sialic acids from glial cells. A recombinant neuraminidase that specifically removes the α(2-3) linkage of sialic acid had no effect on virus binding or infection. A competition assay between virus and sialic acid-specific lectins that recognize either the α(2-3) or the α(2-6) linkage revealed that JCV preferentially interacts with α(2-6)-linked sialic acids on glial cells. Treatment of glial cells with tunicamycin, but not with benzyl N-acetyl-α-d-galactosaminide, inhibited infection by JCV, indicating that the sialylated JCV receptor is an N-linked glycoprotein. As sialic acid containing glycoproteins play a fundamental role in mediating many virus-cell and cell-cell recognition processes, it will be of interest to determine what role these receptors play in the pathogenesis of PML.Approximately 70% of the human population worldwide is seropositive for JC virus (JCV). Like other polyomaviruses, JCV establishes a lifelong latent or persistent infection in its natural host (40, 49, 50, 68, 72). Reactivation of JCV in the setting of an underlying immunosuppressive illness, such as AIDS, is thought to lead to virus dissemination to the central nervous system (CNS) and subsequent infection of oligodendrocytes (37, 40, 66, 68). Reactivation of latent JCV genomes already present in the CNS has also been postulated to contribute to the development of progressive multifocal leukoencephalopathy (PML) following immunosuppression (19, 48, 55, 70, 75). Approximately 4 to 6% of AIDS patients will develop PML during the course of their illness (10). In the CNS, JCV specifically infects oligodendrocytes and astrocytes. Outside the CNS, JCV genomes have been identified in the urogenital system, in the lymphoid system, and in B lymphocytes (2, 17, 18, 30, 47, 59). In vitro, JCV infects human glial cells and, to a limited extent, human B lymphocytes (3, 4, 39, 41, 42). Recently, JCV infection of tonsillar stromal cells and CD34⁺ B-cell precursors has been described (47). These observations have led to the suggestion that JCV may persist in a lymphoid compartment and that B cells may play a role in trafficking of JCV to the CNS (4, 30, 47).Virus-receptor interactions play a major role in determining virus tropism and tissue-specific pathology associated with virus infection. Viruses that have a very narrow host range and tissue tropism, such as JCV, are often shown to interact with high affinity to a limited number of specific receptors present on susceptible cells (26, 44). In some instances, virus tropism is strictly determined by the presence of specific receptors that mediate binding and entry (7, 16, 27, 35, 46, 53, 56, 67, 73, 74, 76). In other instances, however, successful entry into a cell is necessary but not sufficient for virus growth (5, 8, 45, 57). In these cases, additional permissive factors that interact with viral regulatory elements are required.The receptor binding characteristics of several polyomaviruses have been described. The mouse polyomavirus (PyV) receptor is an N-linked glycoprotein containing terminal α(2-3)-linked sialic acid (12–14, 22, 28). Both the large and small plaque strains of PyV recognize α(2-3)-linked sialic acid. The small-plaque strain also recognizes a branched disialyl structure containing α(2-3)- and α(2-6)-linked sialic acids. Neither strain recognizes straight-chain α(2-6)-linked sialic acid. The ability of the large- and small-plaque strains of PyV to differentially recognize these sialic acid structures has been precisely mapped to a single amino acid in the major virus capsid protein VP1 (21). The large-plaque strains all contain a glycine at amino acid position 92 in VP1, and the small-plaque strains all contain a negatively charged glutamic acid at this position (21). In addition to forming small or large plaques, these strains also differ in the ability to induce tumors in mice (20). This finding suggests that receptor recognition plays an important role in the pathogenesis of PyV.The cell surface receptor for lymphotropic papovavirus (LPV) is an O-linked glycoprotein containing terminal α(2-6)-linked sialic acid (26, 33, 34). Infection with LPV is restricted to a subset of human B-cell lines, and recognition of specific receptors is a major determinant of the tropism of LPV for these cells (26).Unlike the other members of the polyomavirus family, infection of cells by simian virus 40 (SV40) is independent of cell surface sialic acids. Instead, SV40 infection is mediated by major histocompatibility complex (MHC)-encoded class I proteins (5, 11). MHC class I proteins also play a role in mediating the association of SV40 with caveolae, a prerequisite for successful targeting of the SV40 genome to the nucleus of a cell (1, 63). Not surprisingly, SV40 has been shown not to compete with the sialic acid-dependent polyomaviruses for binding to host cells (15, 26, 38, 58).Very little is known about the early steps of JCV binding to and infection of glial cells. Like other members of the polyomavirus family, JCV is known to interact with cell surface sialic acids (51, 52). A role for sialic acids in mediating infection of glial cells has not been described. It is also not known whether the sialic acid is linked to a glycoprotein or a glycolipid. In a previous report, we demonstrated that JCV bound to a limited number of cell surface receptors on SVG cells that were not shared by the related polyomavirus SV40 (38). In this report, we demonstrate that virus binding to and infection of SVG cells is dependent on an N-linked glycoprotein containing terminal α(2-3)- and α(2-6)-linked sialic acids. Competitive binding assays with sialic acid-specific lectins suggest that the virus preferentially interacts with α(2-6)-linked sialic acids. We are currently evaluating the role of this receptor in determining the tropism of JCV for glial cells and B cells.     

Dominant-Negative Mutations in the G-Protein-Coupled α-Factor Receptor Map to the Extracellular Ends of the Transmembrane Segments

G-protein-coupled receptors (GPCRs) transduce the signals for a wide range of hormonal and sensory stimuli by activating a heterotrimeric guanine nucleotide-binding protein (G protein). The analysis of loss-of-function and constitutively active receptor mutants has helped to reveal the functional properties of GPCRs and their role in human diseases. Here we describe the identification of a new class of mutants, dominant-negative mutants, for the yeast G-protein-coupled α-factor receptor (Ste2p). Sixteen dominant-negative receptor mutants were isolated based on their ability to inhibit the response to mating pheromone in cells that also express wild-type receptors. Detailed analysis of two of the strongest mutant receptors showed that, unlike other GPCR interfering mutants, they were properly localized at the plasma membrane and did not alter the stability or localization of wild-type receptors. Furthermore, their dominant-negative effect was inversely proportional to the relative amount of wild-type receptors and was reversed by overexpressing the G-protein subunits, suggesting that these mutants compete with the wild-type receptors for the G protein. Interestingly, the dominant-negative mutations are all located at the extracellular ends of the transmembrane segments, defining a novel region of the receptor that is important for receptor signaling. Altogether, our results identify residues of the α-factor receptor specifically involved in ligand binding and receptor activation and define a new mechanism by which GPCRs can be inactivated that has important implications for the evaluation of receptor mutations in other G-protein-coupled receptors.G-protein-coupled receptors (GPCRs) comprise a large family of receptors that are found in a wide range of eukaryotic organisms from yeasts to humans (4, 10). These receptors respond to diverse stimuli including hormones, neurotransmitters, and other chemical messengers (48). GPCRs transduce their signal by stimulating the α subunit of a heterotrimeric guanine nucleotide binding protein (G protein) to bind GTP (4, 16). This releases the α subunit from the βγ subunits, and then either the α subunit or the βγ subunits go on to promote signaling depending on the specific pathway (28).GPCRs are structurally similar in that they contain seven transmembrane domains (TMDs) connected by intracellular and extracellular loops. Although many techniques have been applied to study receptor function, much of our knowledge on the mechanisms of GPCR activation comes from the characterization of mutant receptors. Loss-of-function and supersensitive mutants have helped to identify receptor regions needed for ligand binding, G-protein activation, and down-regulation of signaling (4, 49). Furthermore, the study of constitutively active receptor mutations has played a key role in the development of current models for receptor activation (26). Naturally occurring GPCR mutations have also been implicated in a number of human diseases (8, 25, 42). Interestingly, the analysis of different mutant receptors indicates that GPCRs utilize common structural domains for similar functions. In particular, the third intracellular loop has an essential role in G-protein activation in a wide range of GPCRs.The genetic approaches possible in the yeast Saccharomyces cerevisiae have been used to examine the relationship between structure and function of the G-protein-coupled mating pheromone receptors. The α-factor and a-factor pheromones induce conjugation in yeast by binding to receptors with seven TMDs that activate a G-protein signal pathway that is highly conserved with mammalian signaling pathways (24). In fact, some human GPCRs can activate the pheromone signal pathway when they are expressed in yeast (19, 29). The analysis of loss-of-function, supersensitive, and constitutively active α-factor receptor mutants has begun to reveal the mechanisms for activation and regulation of this receptor. For example, the analysis of constitutively active mutants indicates that movement in the transmembrane segments plays a key role in α-factor receptor activation (22). Constitutive mutations and loss-of-function mutations implicate the third intracellular loop in G-protein activation (7, 34, 44). Mutagenesis studies also indicate that the cytoplasmic C terminus is not needed for G-protein activation but is involved in down-regulation of receptors by endocytosis (17) and desensitization of receptors by phosphorylation (6). In addition, studies with chimeric receptors suggest that the specificity for α-factor binding is determined by discontinuous segments of the α-factor receptor that include the transmembrane and extracellular regions (36, 37). Although some of the important domains of the α-factor receptor have been identified in these studies, the molecular mechanism of receptor signaling remains to be determined.Dominant-negative (DN) mutants represent an important class of mutation in which a mutant receptor interferes with the function of the wild-type (WT) version of the receptor. Since the inhibitory phenotype in DN mutants implies loss of some but not all functions of the protein, these mutants have been used to great advantage in other receptor systems. For example, in the case of receptor tyrosine kinases, DN mutants have been used to assign particular functions to specific structural features or to study the effects of blocking receptor signaling (18). In view of the large number of mutations reported for GPCRs, it is intriguing that there are few examples of dominant GPCR mutations (42, 43). Furthermore, in cases where it has been examined, dominant mutations in GPCRs seem to affect primarily the targeting of receptors to the plasma membrane and not directly the function of the WT receptors. Therefore, we sought to determine if the analysis of DN mutants could be applied to GPCRs by taking advantage of the genetic accessibility of the yeast S. cerevisiae. In this report, we describe the identification of DN mutations in the α-factor pheromone receptor. Interestingly, our results indicate that these DN mutants interfere with the activity of the WT receptors by competing for the G protein. In addition, these mutations identify a new domain on the extracellular side of the TMDs that is important for receptor function.