Activation of a G-protein-coupled receptor involves changes in specific microdomain interactions within the transmembrane region of the receptor. Here, we have focused on the role of L207, proximal to the DRY motif of the human cannabinoid receptor 1 in the interconversion of the receptor resting and active states. Ligand binding analysis of the mutant receptor L207A revealed an enhanced affinity for agonists (three- to six-fold) and a diminished affinity for inverse agonists (19- to 35-fold) compared to the wild-type receptor, properties characteristic of constitutive activity. To further examine whether this mutant adopts a ligand-independent, active form, treatment with GTPγS was used to inhibit G protein coupling. Under these conditions, the L207A receptor exhibited a 10-fold increase in affinity for the inverse agonist SR141716A, consistent with a shift away from an enhanced precoupled state. Analysis of the cellular activity of the L207A receptor showed elevated basal cyclic AMP accumulation relative to the wild type that is inhibited by SR141716A, consistent with receptor-mediated Gs precoupling. Using toxins to selectively abrogate Gs or Gi coupling, we found that CP55940 nonetheless induced only a Gi response suggesting a strong preference of this ligand-bound form for Gi in this system. Molecular dynamics simulations reveal that the single residue change of L207A impacts the association of TM3 and TM6 in the receptor by altering hydrophobic interactions involving L207, the salt bridge involving the Arg of the DRY motif, and the helical structure of TM6, consistent with events leading to activation. The structural alterations parallel those observed in models of a mutant CB1 receptor T210I, with established constitutive activity (D’Antona, A.M., Ahn, K.H. and Kendall, D.A., 2006. Mutations of CB1 T210 produce active and inactive receptor forms: correlations with ligand affinity, receptor stability, and cellular localization. Biochemistry, 45, 5606–5617).
Cannabinoid; Cannabinoid receptor; CB1; G-protein-coupled receptor; Ligand binding; Receptor activation
Genes encoding chemokine receptor-like proteins have been found in
herpes and poxviruses and implicated in viral pathogenesis. Here we
describe the cellular distribution and trafficking of a human
cytomegalovirus (HCMV) chemokine receptor encoded by the
US28 gene, after transient and stable expression in
transfected HeLa and Cos cells. Immunofluorescence staining indicated
that this viral protein accumulated intracellularly in vesicular
structures in the perinuclear region of the cell and showed overlap
with markers for endocytic organelles. By immunogold electron
microscopy US28 was seen mostly to localize to multivesicular
endosomes. A minor portion of the protein (at most 20%) was also
expressed at the cell surface. Antibody-feeding experiments indicated
that cell surface US28 undergoes constitutive ligand-independent
endocytosis. Biochemical analysis with the use of iodinated ligands
showed that US28 was rapidly internalized. The high-affinity ligand of
US28, the CX3C-chemokine fractalkine, reduced the
steady-state levels of US28 at the cell surface, apparently by
inhibiting the recycling of internalized receptor. Endocytosis and
cycling of HCMV US28 could play a role in the sequestration of host
chemokines, thereby modulating antiviral immune responses. In addition,
the distribution of US28 mainly on endosomal membranes may allow it to
be incorporated into the viral envelope during HCMV assembly.
The viral G-protein coupled receptor (vGPCR) specified by human herpesvirus 8 (HHV-8) open reading frame 74 (ORF74) is a ligand-independent chemokine receptor that has structural and functional homologues among other characterized gammaherpesviruses and related receptors in the betaherpesviruses. Sequence comparisons of the gammaherpesvirus vGPCRs revealed a highly conserved region in the C tail, just distal to the seventh transmembrane domain. Mutagenesis of the corresponding codons of HHV-8 ORF74 was carried out to provide C-tail-altered proteins for functional analyses. By measuring receptor-activated vascular endothelial growth factor promoter induction and NF-κB, mitogen-activated protein kinase, and Ca2+ signaling, we found that while some altered receptors showed general signaling deficiencies, others had distinguishable activation profiles, suggestive of selective Gα protein coupling. This was supported by the finding that vGPCR and representative functionally altered variants, vGPCR.8 (R322W) and vGPCR.15 (M325S), were affected differently by inhibitors of Gαi (pertussis toxin), protein kinase C (GF109203X), and phosphatidylinositol 3-kinase (wortmannin). Consistent with the signaling data, [35S]GTPγS incorporation assays revealed preferential coupling of vGPCR.15 to Gαq and an inability of vGPCR.8 to couple functionally to Gαq. However, both variants, wild-type vGPCR, and a C-tail deletion version of the receptor were equally able to associate physically with Gαq. Combined, our data demonstrate that HHV-8 vGPCR contains discrete sites of Gα interaction and that receptor residues in the proximal region of the cytoplasmic tail are determinants of Gα protein coupling specificity.
The sequential interaction of the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) with CD4 and certain chemokine coreceptors initiates host cell entry of the virus. The appropriate chemokines have been shown to inhibit viral replication by blocking interaction of the gp120 envelope protein with the coreceptors. We considered the possibility that this interaction involves a motif of the gp120 that may be structurally homologous to the chemokines. In the amino acid sequences of most chemokines there is a Trp residue located at the beginning of the C-terminal α-helix, which is separated by six residues from the fourth Cys residue. The gp120 of all HIV-1 isolates have a similar motif, which includes the C-terminal part of a variable loop 3 (V3) and N-terminal part of a conserved region 3 (C3). Two synthetic peptides, derived from the relevant gp120 sequence inhibited HIV-1 replication in macrophages and T lymphocytes in sequence-dependent manner. The peptides also prevented binding of anti-CXCR4 antibodies to CXCR4, and inhibited the intracellular Ca2+ influx in response to CXCL12/SDF-1α. Thus these peptides can be used to dissect gp120 interactions with chemokine receptors and could serve as leads for the design of new inhibitors of HIV-1.
Natural killer cells express clonally distributed receptors specific for major histocompatibility complex class I molecules. The human leukocyte antigen (HLA)-C-specific receptors have been molecularly identified and cloned. They exist not only as inhibitory (p58) but also as activatory (p50) receptors. Here we show that p50 and p58 are highly homologous in their extracellular regions formed by two Ig-like domains. In contrast, major differences exist in their transmembrane and cytoplasmic portions. Whereas p 58 displays a 76-84-amino acid cytoplasmic tail containing an unusual antigen receptor activation motif, p50 is characterized by a shorter 39-amino acid tail. In addition, whereas p58 has a nonpolar transmembrane portion, p50 contains the charged amino acid Lys. These data strongly suggest that receptors with identical HLA-C allele specificity can mediate functions of opposite sign owing to their different transmembrane/cytoplasmic portions.
To complete their life cycle and spread, viruses interfere with and gain control of diverse cellular processes, this most often occurring through interaction between viral proteins (VPs) and resident protein partners. Among the latter, Src family kinases (SFKs), a class of non-receptor tyrosine kinases that contributes to the conversion of extracellular signals into intracellular signaling cascades and is involved in virtually all cellular processes, have recently emerged as critical mediators between the cell’s infrastructure and the viral demands. In this scenario, structural or ex novo synthesized VPs are able to bind to the different domains of these enzymes through specific short linear motifs present along their sequences. Proline-rich motifs displaying the conserved minimal consensus PxxP and recognizing the SFK Src homology (SH)3 domain constitute a cardinal signature for the formation of multiprotein complexes and this interaction may promote phosphorylation of VPs by SFKs, thus creating phosphotyrosine motifs that become a docking site for the SH2 domains of SFKs or other SH2 domain-bearing signaling molecules. Importantly, the formation of these assemblies also results in a change in the activity and/or location of SFKs, and these events are critical in perturbing key signaling pathways so that viruses can utilize the cell’s machinery to their own benefit. In the light of these observations, although VPs as such, especially those with enzyme activity, are still regarded as valuable targets for therapeutic strategies, multiprotein complexes composed of viral and host cell proteins are increasingly becoming objects of investigation with a view to deeply characterize the structural aspects that favor their formation and to develop new compounds able to contrast viral diseases in an alternative manner.
Interaction; Phosphotyrosine; Proline-rich motif; Src homology 2 domain; Src homology 3 domain
The heptad repeat (HR), a conserved structural motif of class I viral fusion proteins, is responsible for the formation of a six-helix bundle structure during the envelope fusion process. The insect baculovirus F protein is a newly found budded virus envelope fusion protein which possesses common features to class I fusion proteins, such as proteolytic cleavage and the presence of an N-terminal open fusion peptide and multiple HR domains on the transmembrane subunit F1. Similar to many vertebrate viral fusion proteins, a conserved leucine zipper motif is predicted in this HR region proximal to the fusion peptide in baculovirus F proteins. To facilitate our understanding of the functional role of this leucine zipper-like HR1 domain in baculovirus F protein synthesis, processing, and viral infectivity, key leucine residues (Leu209, Leu216, and Leu223) were replaced by alanine (A) or arginine (R), respectively. By using Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) as a pseudotype expression system, we demonstrated that all mutant F proteins incorporated into budded virus, indicating that leucine substitutions did not affect intercellular trafficking of F. Furin-like protease cleavage was not affected by any of the leucine substitutions; however, the disulfide bridging and N-linked glycosylation patterns were partly altered. Single substitutions in HR1 showed that the three leucine residues were critical for F fusogenicity and the rescue of AcMNPV infectivity. Our results support the view that the leucine zipper-like HR1 domain is important to safeguard the proper folding, glycosylation, and fusogenicity of baculovirus F proteins.
Programmed cell death signaling is a critical feature of development, cellular turnover, oncogenesis, and neurodegeneration, among other processes. Such signaling may be transduced via specific receptors, either following ligand binding—to death receptors—or following the withdrawal of trophic ligands—from dependence receptors. Although dependence receptors display functional similarities, no common structural domains have been identified. Therefore, we employed the Multiple Expectation Maximization for Motif Elicitation and the Motif Alignment and Search Tool software programs to identify a novel transmembrane motif, dubbed dependence-associated receptor transmembrane (DART) motif, that is common to all described dependence receptors. Of 3,465 human transmembrane proteins, 25 (0.7%) display the DART motif. The predicted secondary structure features an alpha helical structure, with an unusually high percentage of valine residues. At least four of the proteins undergo regulated intramembrane proteolysis. To date, we have not identified a function for this putative domain. We speculate that the DART motif may be involved in protein processing, interaction with other proteins or lipids, or homomultimerization.
The carboxyl tail of G protein-coupled receptors contains motifs that regulate receptor interactions with intracellular partners. Activation of the human neutrophil complement fragment C5a receptor (C5aR) is terminated by phosphorylation of the carboxyl tail followed by receptor internalization. In this study, we demonstrated that bulky hydrophobic residues in the membrane proximal region of the C5aR carboxyl tail play an important role in proper structure and function of the receptor: Substitution of leucine 319 with alanine (L319A) resulted in receptor retention in the endoplasmic reticulum, whereas a L318A substitution allowed receptor transport to the cell surface, but showed slow internalization upon activation, presumably due to a defect in phosphorylation by both PKC and GRK. Normal agonist-induced activation of ERK1/2 and intracellular calcium release suggested that the L318A mutation did not affect receptor signaling. Binding of GRK2 and PKCβII to intracellular loop 3 of C5aR in vitro indicated that mutagenesis of L318 did not affect kinase binding. Limited proteolysis with trypsin revealed a conformational difference between wild type and mutant receptor. Our studies support a model in which the L318/L319 stabilizes an amphipathic helix (Q305–R320) in the membrane proximal region of C5aR.
Chemoattractant receptor; endocytosis; protein folding; cytoplasmic helix 8
The highly conserved DRY motif located at the end of the third transmembrane of G protein-coupled receptors has been described as a key motif for several aspects of GPCR functions. However, in the case of the vertebrate gonadotropin-releasing hormone receptor (GnRHR), the amino acid in the third position in the DRY motif is variable. In the lamprey, a most basal vertebrate, the third amino acid of the “DRY” in GnRHR is His, while it is most often His/Gln in the type II GnRHR. To investigate the functional significance of the substitution of DRY to DRH in the lamprey(l)GnRHR, second messenger signaling, ligand binding and internalization of the wild-type and mutant lGnRH receptors were characterized with site-directed mutagenesis. Treatment of the DRE151 and DRS151 mutant receptors with lamprey GnRH-I significantly reduced inositol phosphate compared to wild-type (DRH151) and DRY151 receptors. The logIC50 of wild-type receptor (−9.554±0.049) was similar to the logIC50 of DRE151, DRS151 and DRX151 mutants, yet these same mutants were shown to significantly reduce cell surface expression. However, the DRY151 mutant compared to the wild-type receptor increased cell surface expression, suggesting that the reduction of IP production was due to the level of the cell surface expression of the mutant receptors. The rate of internalization of DRX151 (35.60%) was reduced compared to wild-type and other mutant receptors. These results suggest that His151 of the lamprey GnRH receptor may play a critical role in the retention of a certain level of cell-surface expression for subsequent cellular second messenger events.
GnRH receptor; DRY motif; receptor expression; signaling; site-directed mutagenesis; lamprey
Toll like receptors (TLRs) are essential for host defense. While several TLRs reside on the cell surface, nucleic acid recognizing TLRs are intracellular. For example, the receptor for CpG containing bacterial and viral DNA, TLR9, is retained in the endoplasmic reticulum (ER). Recent evidence suggests that the localization of TLR9 is critical for appropriate ligand recognition. Here we define which structural features of the TLR9 molecule control its intracellular localization. Both the cytoplasmic and ectodomains of TLR9 contain sufficient information while the transmembrane domain plays no role in intracellular localization. We identify a 14 amino acid stretch that directs TLR9 intracellularly and confers intracellular localization to the normally cell surface expressed TLR4. Truncation or mutation of the cytoplasmic tail of TLR9 reveals a vesicle localization motif that targets early endosomes. We propose a model whereby modification of the cytoplasmic tail of TLR9 results in trafficking to early endosomes where it encounters CpG DNA.
Chemokines and their receptors play important roles in host defense, organogenesis, hematopoiesis, and neuronal communication. Forty-two chemokines and 19 cognate receptors have been found in the human genome. Prior to this report, only 11 chicken chemokines and 7 receptors had been reported. The objectives of this study were to systematically identify chicken chemokines and their cognate receptor genes in the chicken genome and to annotate these genes and ligand-receptor binding by a comparative genomics approach.
Twenty-three chemokine and 14 chemokine receptor genes were identified in the chicken genome. All of the chicken chemokines contained a conserved CC, CXC, CX3C, or XC motif, whereas all the chemokine receptors had seven conserved transmembrane helices, four extracellular domains with a conserved cysteine, and a conserved DRYLAIV sequence in the second intracellular domain. The number of coding exons in these genes and the syntenies are highly conserved between human, mouse, and chicken although the amino acid sequence homologies are generally low between mammalian and chicken chemokines. Chicken genes were named with the systematic nomenclature used in humans and mice based on phylogeny, synteny, and sequence homology.
The independent nomenclature of chicken chemokines and chemokine receptors suggests that the chicken may have ligand-receptor pairings similar to mammals. All identified chicken chemokines and their cognate receptors were identified in the chicken genome except CCR9, whose ligand was not identified in this study. The organization of these genes suggests that there were a substantial number of these genes present before divergence between aves and mammals and more gene duplications of CC, CXC, CCR, and CXCR subfamilies in mammals than in aves after the divergence.
The cannabinoid type-1 (CB1) receptor is a G protein-coupled receptor (GPCR) that binds the main active ingredient of marijuana, Δ9-tetrahydrocannabinol, and has been implicated in several disease states, including drug addiction, anxiety, depression, obesity, and chronic pain. In the two decades since the discovery of CB1, studies at the molecular level have centered on the transmembrane core. This interest has now expanded as we discover that other regions of CB1, including the CB1 carboxyl-terminus, have critical structures that are important for CB1 activity and regulation. Following the recent description of the three dimensional structure of the full-length CB1 carboxyl-terminal tail (Ahn et al., Biopolymers (2009) 91: 565–573), several residues and structural motifs including two α-helices (termed H8 and H9) have been postulated to interact with common GPCR accessory proteins, such as G-proteins and β-arrestins. This discourse will focus on the CB1 carboxyl-terminus; our current understanding of the structural features of this region, evidence for its interaction with proteins, and the impact of structure on the binding and regulatory function of CB1 accessory proteins. The involvement of the carboxyl-terminus in the receptor life cycle including activation, desensitization, and internalization will be highlighted.
cannabinoid receptor; G protein-coupled receptor; carboxyl-terminus; internalization; desensitization; helix 8
Simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) Nef proteins are related regulatory proteins that share several functions, including the ability to downregulate class I major histocompatibility complex (MHC) and CD4 expression on the cell surface and to alter T-cell-receptor-initiated signal transduction in T cells. We compared the mechanisms used by SIV mac239 Nef and HIV-1 Nef to downregulate class I MHC and found that the ability of SIV Nef to downregulate class I MHC requires a unique C-terminal region of the SIV mac239 Nef molecule which is not found in HIV-1 Nef. Interestingly, mutation of the PxxP motif in SIV Nef, unlike in HIV-1 Nef, does not affect class I MHC downregulation. We also found that downregulation of class I MHC by SIV Nef requires a conserved tyrosine in the cytoplasmic domain of the class I MHC heavy chain and involves accelerated endocytosis of class I complexes, as previously found with HIV-1 Nef. Thus, while SIV and HIV-1 Nef proteins use a similar mechanism to downregulate class I MHC expression, they have evolved different surfaces for molecular interactions with cell factors that regulate class I MHC traffic. Mutations in the C-terminal domain of SIV mac239 Nef selectively disrupt class I MHC downregulation, having no detectable effect on other functions of Nef, such as the downregulation of CD4 and CD3 surface expression, the stimulation of SIV virion infectivity, and the induction of SIV replication from T cells infected in the absence of stimulation. The resulting mutants will be useful reagents for studying the importance of class I MHC downregulation for SIV replication and AIDS pathogenesis in infected rhesus macaques.
Human androgen receptor (AR) transcriptional activity involves interdomain and coactivator interactions with the agonist-bound AR ligand binding domain (LBD). Structural determinants of the AR NH2- and carboxyl-terminal interaction between the AR NH2-terminal FXXLF motif and activation function 2 (AF2) in the LBD were shown previously by crystallography. In this report, we provide evidence for a region in AR LBD helix 12 outside the AF2 binding cleft that facilitates interactions with the FXXLF and LXXLL motifs. Mutagenesis of glutamine 902 to alanine in AR LBD helix 12 (Q902A) disrupted AR FXXLF motif binding to AF2, but enhanced coactivator LXXLL motif binding. Functional compensation for defective FXXLF motif binding by AR-Q902A was suggested by the slower dissociation rate of bound androgen. Functional importance of glutamine 902 was indicated by the charged residue germline mutation Q902R that caused partial androgen insensitivity, and a similar somatic mutation Q902K reported in prostate cancer, both of which increased the androgen dissociation rate and decreased AR transcriptional activity. High affinity equilibrium androgen binding was retained by alanine substitution mutations at Tyr-739 in AR LBD helix 5 or Lys-905 in helix 12 structurally adjacent to AF2, whereas transcriptional activity decreased and the androgen dissociation increased. Deleterious effects of these loss of function mutations were rescued by the helix stabilizing AR prostate cancer somatic mutation H874Y. Sequence NH2-terminal to the AR FXXLF motif contributed to the AR NH2- and carboxyl-terminal interaction based on greater AR-2-30 FXXLF motif peptide binding to the agonist-bound AR LBD than a shorter AR-20-30 FXXLF motif peptide. We conclude that helix 12 residues outside the AF2 binding cleft modulate AR transcriptional activity by providing flexibility to accommodate FXXLF or LXXLL motif binding.
androgen receptor; N/C interaction; FXXLF motif; LXXLL motif; androgen insensitivity; prostate cancer
The design and development of selective ligands for the human OT (oxytocin) and AVP (arginine vasopressin) receptors is a big challenge since the different receptor subtypes and their native peptide ligands display great similarity. Detailed understanding of the mechanism of OT's interaction with its receptor is important and may assist in the ligand- or structure-based design of selective and potent ligands. In the present article, we compared 69 OT- and OT-like receptor sequences with regards to their molecular evolution and diversity, utilized an in silico approach to map the common ligand interaction sites of recently published G-protein-coupled receptor structures to a model of the human OTR (OT receptor) and compared these interacting residues within a selection of different OTR sequences. Our analysis suggests the existence of a binding site for OT peptides within the common transmembrane core region of the receptor, but it appears extremely difficult to identify receptor or ligand residues that could explain the selectivity of OT to its receptors. We remain confident that the presented evolutionary overview and modelling approach will aid interpretation of forthcoming OTR crystal structures.
arginine vasopressin; binding; GPCR (G-protein-coupled receptor); homology model; OT (oxytocin); vasotocin; AVP, arginine vasopressin; CTR, cephalotocin receptor; GPCR, G-protein-coupled receptor; ICL, intracellular loop; OT, oxytocin; OTR, oxytocin receptor; TM, transmembrane; VT, vasotocin
Terminal tetraloops consisting of GNRA sequences are often found in biologically active large RNAs. The loops appear to contribute towards the organization of higher order RNA structures by forming specific tertiary interactions with their receptors. Group IC3 introns which possess a GAAA loop in the L2 region often have a phylogenetically conserved motif in their P8 domains. In this report, we show that this conserved motif stands as a new class of receptor that distinguishes the sequences of GNRA loops less stringently than previously known receptors. The motif can functionally substitute an 11 nt motif receptor in the Tetrahymena ribozyme. Its structural and functional similarity to one class of synthetic receptors obtained from in vitro selection is observed.
The mammalian olfactory apparatus is able to recognize and distinguish thousands of structurally diverse volatile chemicals. This chemosensory function is mediated by a very large family of seven-transmembrane olfactory (odorant) receptors encoded by approximately 1,000 genes, the majority of which are believed to be pseudogenes in humans.
The strategy of our sequence database mining for full-length, functional candidate odorant receptor genes was based on the high overall sequence similarity and presence of a number of conserved sequence motifs in all known mammalian odorant receptors as well as the absence of introns in their coding sequences. We report here the identification and physical cloning of 347 putative human full-length odorant receptor genes. Comparative sequence analysis of the predicted gene products allowed us to identify and define a number of consensus sequence motifs and structural features of this vast family of receptors. A new nomenclature for human odorant receptors based on their chromosomal localization and phylogenetic analysis is proposed. We believe that these sequences represent the essentially complete repertoire of functional human odorant receptors.
The identification and cloning of all functional human odorant receptor genes is an important initial step in understanding receptor-ligand specificity and combinatorial encoding of odorant stimuli in human olfaction.
The activity of human immunodeficiency virus Rev as a regulator of viral mRNA expression is tightly linked to its ability to shuttle between the nucleus and cytoplasm; these properties are conferred by a leucine-rich nuclear export signal (NES) and by an arginine-rich nuclear localization signal/RNA binding domain (NLS/RBD) required for binding to the Rev-responsive element (RRE) located on viral unspliced and singly spliced mRNAs. Structure predictions and biophysical measurements indicate that Rev consists of an unstructured region followed by a helix-loop-helix motif containing the NLS/RBD and sequences directing multimerization and by a carboxy-terminal tail containing the NES. We present evidence that the loop portion of the helix-loop-helix region is an essential functional determinant that is required for binding to the RRE and for correct intracellular routing. Data obtained using a protein kinase CK2 phosphorylation assay indicated that the loop region is essential for juxtaposition of helices 1 and 2 and phosphorylation by protein kinase CK2. Deletion of the loop resulted in partial accumulation of Rev in SC35-positive nuclear bodies that resembled nuclear bodies that form in response to inhibition of transcription. Accumulation of the ΔLoop mutant in nuclear bodies depended on the presence of an intact NES, suggesting that both the loop and the NES play a role in controlling intranuclear compartmentalization of Rev and its association with splicing factors.
Purpose of review
Class A G protein-coupled receptors, including the chemokine receptors, CCR5 and CXCR4, share a seven transmembrane-spanning α-helix architecture that accommodates signal propagation from across biological membranes. CXCR4 and CCR5 are utilized as co-receptors during HIV viral entry and therefore crystal structures of GPCRs aid in the understanding of their function in viral entry.
Recent progress in structure determination of class A GPCRs, which include vertebrate and invertebrate rhodopsin as well as adrenergic and adenosine receptors, provide molecular templates for how this diverse group of transmembrane receptors functions. Each of these GPCRs differs in how specific ligands bind to the transmembrane core, underscoring that additional structures of GPCRs from other sub-families are needed to facilitate rational drug design. More recent studies also indicate a need to consider the more complex character of GPCRs, such as their oligomerization and dynamics.
Recently the atomic structures of invertebrate rhodopsin, β1- and β2-adrenergic receptors and the A2A-adenosine receptor have been solved via X-ray crystallography. The impact that these structures have on the biochemistry of viral entry and signal transduction is discussed. Because the chemokine receptors have proven refractory to structural studies thus far, further structural study of the chemokine receptors will be essential to understanding ligand binding, activation and function as co-receptors during viral entry.
rhodopsin; G protein-coupled receptor; membrane protein crystallography; GPCR; CCR5; CXCR4
The E3 ubiquitin ligase atrophin interacting protein 4 (AIP4) mediates ubiquitination and down-regulation of the chemokine receptor CXCR4. AIP4 belongs to the Nedd4-like homologous to E6-AP carboxy terminus domain family of E3 ubiquitin ligases, which typically bind proline-rich motifs within target proteins via the WW domains. The intracellular domains of CXCR4 lack canonical WW domain binding motifs; thus, whether AIP4 is targeted to CXCR4 directly or indirectly via an adaptor protein remains unknown. Here, we show that AIP4 can interact directly with CXCR4 via a novel noncanonical WW domain-mediated interaction involving serine residues 324 and 325 within the carboxy-terminal tail of CXCR4. These serine residues are critical for mediating agonist-promoted binding of AIP4 and subsequent ubiquitination and degradation of CXCR4. These residues are phosphorylated upon agonist activation and phosphomimetic mutants show enhanced binding to AIP4, suggesting a mechanism whereby phosphorylation mediates the interaction between CXCR4 and AIP4. Our data reveal a novel noncanonical WW domain-mediated interaction involving phosphorylated serine residues in the absence of any proline residues and suggest a novel mechanism whereby an E3 ubiquitin ligase is targeted directly to an activated G protein-coupled receptor.
Lgr5 is a membrane protein related to G protein-coupled receptors (GPCR)s whose expression identifies stem cells in multiple tissues and is strongly correlated with cancer. Despite the recent identification of endogenous ligands for Lgr5, its mode of signaling remains enigmatic. The ability to couple to G proteins and βarrestins are classical molecular behaviors of GPCRs that have yet to be observed for Lgr5. Therefore, the goal of this study was to determine if Lgr5 can engage a classical GPCR behavior and elucidate the molecular determinants of this process. Structural analysis of Lgr5 revealed several motifs consistent with its ability to recruit βarr2. Among them, a “SSS” serine cluster located at amino acid position 873-875 within the C-terminal tail (C-tail), is in a region consistent with other GPCRs that bind βarr2 with high-affinity. To test its functionality, a ligand-independent βarr2 translocation assay was implemented. We show that Lgr5 recruits βarr2 and that the “SSS” amino acids (873-875) are absolutely critical to this process. We also demonstrate that for full efficacy, this cluster requires other Lgr5 C-tail serines that were previously shown to be important for constitutive and βarr2 independent internalization of Lgr5. These data are proof of principle that a classical GPCR behavior can be manifested by Lgr5. The existence of alternative ligands or missing effectors of Lgr5 that scaffold this classical GPCR behavior and the downstream signaling pathways engaged should be considered. Characterizing Lgr5 signaling will be invaluable for assessing its role in tissue maintenance, repair, and disease.
From computational simulations of a serotonin 2A receptor (5-HT2AR) model complexed with pharmacologically and structurally diverse ligands we identify different conformational states and dynamics adopted by the receptor bound to the full agonist 5-HT, the partial agonist LSD, and the inverse agonist Ketanserin. The results from the unbiased all-atom molecular dynamics (MD) simulations show that the three ligands affect differently the known GPCR activation elements including the toggle switch at W6.48, the changes in the ionic lock between E6.30 and R3.50 of the DRY motif in TM3, and the dynamics of the NPxxY motif in TM7. The computational results uncover a sequence of steps connecting these experimentally-identified elements of GPCR activation. The differences among the properties of the receptor molecule interacting with the ligands correlate with their distinct pharmacological properties. Combining these results with quantitative analysis of membrane deformation obtained with our new method (Mondal et al, Biophysical Journal 2011), we show that distinct conformational rearrangements produced by the three ligands also elicit different responses in the surrounding membrane. The differential reorganization of the receptor environment is reflected in (i)-the involvement of cholesterol in the activation of the 5-HT2AR, and (ii)-different extents and patterns of membrane deformations. These findings are discussed in the context of their likely functional consequences and a predicted mechanism of ligand-specific GPCR oligomerization.
The 5-HT2A receptor for the neurotransmitter serotonin (5-HT) belongs to family A (rhodopsin-like) G-protein coupled receptors (GPCRs), one of the most important classes of membrane proteins that are targeted by an extensive and diverse collection of external stimuli. Recently we learned that different ligands targeting the same GPCR can elicit different biological responses, but the mechanisms remain unknown. We address this fundamental question for the serotonin 5-HT2A receptor, because it is known to respond to the binding of structurally diverse ligands by producing similar stimuli in the cell, and to the binding of quite similar ligands with dramatically different responses. Molecular dynamics simulations of molecular models of the serotonin 5-HT2A receptor in complex with pharmacologically distinct ligands show the dynamic rearrangements of the receptor molecule to be different for these ligands, and the nature and extents of the rearrangements reflect the known pharmacological properties of the ligands as full, partial or inverse activators of the receptor. The different rearrangements of the receptor molecule are shown to produce different rearrangements of the surrounding membrane, a remodeling of the environment that can have differential ligand-determined effects on receptor function and association in the cell's membrane.
The chemokine (C-C motif) receptor 2B (CCR2B) is one of the two isoforms of the receptor for monocyte chemoattractant protein-1 (CCL2), the major chemoattractant for monocytes, involved in an array of chronic inflammatory diseases. Employing the yeast two-hybrid system, we identified the actin-binding protein filamin A (FLNa) as a protein that associates with the carboxyl-terminal tail of CCR2B. Co-immunoprecipitation experiments and in vitro pull down assays demonstrated that FLNa binds constitutively to CCR2B. The colocalization of endogenous CCR2B and filamin A was detected at the surface and in internalized vesicles of THP-1 cells. In addition, CCR2B and FLNa were colocalized in lamellipodia structures of CCR2B-expressing A7 cells. Expression of the receptor in filamin-deficient M2 cells together with siRNA experiments knocking down FLNa in HEK293 cells, demonstrated that lack of FLNa delays the internalization of the receptor. Furthermore, depletion of FLNa in THP-1 monocytes by RNA interference reduced the migration of cells in response to MCP-1. Therefore, FLNa emerges as an important protein for controlling the internalization and spatial localization of the CCR2B receptor in different dynamic membrane structures.
RNA molecules take advantage of prevalent structural motifs to fold and assemble into well-defined 3D architectures. The A-minor junction is a class of RNA motifs that specifically controls coaxial stacking of helices in natural RNAs. A sensitive self-assembling supra-molecular system was used as an assay to compare several natural and previously unidentified A-minor junctions by native polyacrylamide gel electrophoresis and atomic force microscopy. This class of modular motifs follows a topological rule that can accommodate a variety of interchangeable A-minor interactions with distinct local structural motifs. Overall, two different types of A-minor junctions can be distinguished based on their functional self-assembling behavior: one group makes use of triloops or GNRA and GNRA-like loops assembling with helices, while the other takes advantage of more complex tertiary receptors specific for the loop to gain higher stability. This study demonstrates how different structural motifs of RNA can contribute to the formation of topologically equivalent helical stacks. It also exemplifies the need of classifying RNA motifs based on their tertiary structural features rather than secondary structural features. The A-minor junction rule can be used to facilitate tertiary structure prediction of RNAs and rational design of RNA parts for nanobiotechnology and synthetic biology.