ABSTRACT
ß-arrestins (ßarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven primarily by agonist-induced receptor activation and phosphorylation. Here, we present seven cryo-electron microscopy structures of ßarrs either in the basal state, activated by the muscarinic receptor subtype 2 (M2R) through its third intracellular loop, or activated by the ßarr-biased decoy D6 receptor (D6R). Combined with biochemical, cellular, and biophysical experiments, these structural snapshots allow the visualization of atypical engagement of ßarrs with 7TMRs and also reveal a structural transition in the carboxyl terminus of ßarr2 from a ß strand to an α helix upon activation by D6R. Our study provides previously unanticipated molecular insights into the structural and functional diversity encoded in 7TMR-ßarr complexes with direct implications for exploring novel therapeutic avenues.
Subject(s)
Protein Interaction Domains and Motifs , Receptors, G-Protein-Coupled , beta-Arrestins , beta-Arrestins/chemistry , Cryoelectron Microscopy , Receptors, G-Protein-Coupled/chemistry , Signal Transduction , Protein Conformation, beta-Strand , Protein Conformation, alpha-Helical , HumansABSTRACT
Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of ß-arrestin (ßarr) recruitment and trafficking. For several GPCRs such as the vasopressin receptor subtype 2 (V2R), agonist-stimulation first drives the translocation of ßarrs to the plasma membrane, followed by endosomal trafficking, which is generally considered to be orchestrated by multiple phosphorylation sites. We have previously shown that mutation of a single phosphorylation site in the V2R (i.e., V2RT360A) results in near-complete loss of ßarr translocation to endosomes despite robust recruitment to the plasma membrane, and compromised ERK1/2 activation. Here, we discover that a synthetic intrabody (Ib30), which selectively recognizes activated ßarr1, efficiently rescues the endosomal trafficking of ßarr1 and ERK1/2 activation for V2RT360A. Molecular dynamics simulations reveal that Ib30 enriches active-like ßarr1 conformation with respect to the inter-domain rotation, and cellular assays demonstrate that it also enhances ßarr1-ß2-adaptin interaction. Our data provide an experimental framework to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can be potentially integrated in the paradigm of GPCR-targeted drug discovery.
Subject(s)
Receptors, G-Protein-Coupled , Signal Transduction , Phosphorylation , Receptors, G-Protein-Coupled/metabolism , beta-Arrestin 1/genetics , beta-Arrestin 1/metabolism , beta-Arrestin 2/metabolism , beta-Arrestins/metabolismABSTRACT
G-protein-coupled receptors (GPCRs), also known as seven transmembrane receptors (7TMRs), typically interact with two distinct signal-transducers, i.e., G proteins and ß-arrestins (ßarrs). Interestingly, there are some non-canonical 7TMRs that lack G protein coupling but interact with ßarrs, although an understanding of their transducer coupling preference, downstream signaling, and structural mechanism remains elusive. Here, we characterize two such non-canonical 7TMRs, namely, the decoy D6 receptor (D6R) and the complement C5a receptor subtype 2 (C5aR2), in parallel with their canonical GPCR counterparts. We discover that D6R and C5aR2 efficiently couple to ßarrs, exhibit distinct engagement of GPCR kinases (GRKs), and activate non-canonical downstream signaling pathways. We also observe that ßarrs adopt distinct conformations for D6R and C5aR2, compared to their canonical GPCR counterparts, in response to common natural agonists. Our study establishes D6R and C5aR2 as ßarr-coupled 7TMRs and provides key insights into their regulation and signaling with direct implication for biased agonism.
Subject(s)
Cell Membrane/metabolism , Protein Conformation , Signal Transduction , beta-Arrestins/chemistry , Animals , GTP-Binding Proteins/chemistry , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , Protein Binding , Protein Domains , Protein Structure, Secondary , Protein Transport , Receptor, Anaphylatoxin C5a/metabolismABSTRACT
Intrauterine bony fragments (IUBF) are an unusual finding in hysterectomy specimen received in a histopathology laboratory. Females harboring IUBF may present non-specific symptoms like vaginal bleeding, leukorrhea, chronic pelvic pain, and secondary infertility. Herein we report the case of a 35-year-old female who presented vaginal discharge and bleeding for two years, since when she had an abortion. Later, hysterectomy specimen revealed bone pieces in the uterine cavity.
ABSTRACT
Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a key determinant for their interaction with ß-arrestins (ßarrs) and subsequent functional responses. Therefore, it is important to decipher the contribution and interplay of different receptor phosphorylation sites in governing ßarr interaction and functional outcomes. Here, we find that several phosphorylation sites in the human vasopressin receptor (V2R), positioned either individually or in clusters, differentially contribute to ßarr recruitment, trafficking, and ERK1/2 activation. Even a single phosphorylation site in V2R, suitably positioned to cross-talk with a key residue in ßarrs, has a decisive contribution in ßarr recruitment, and its mutation results in strong G-protein bias. Molecular dynamics simulation provides mechanistic insights into the pivotal role of this key phosphorylation site in governing the stability of ßarr interaction and regulating the interdomain rotation in ßarrs. Our findings uncover important structural aspects to better understand the framework of GPCR-ßarr interaction and biased signaling.
ABSTRACT
ß-arrestins (ßarrs) are key regulators of G protein-coupled receptor (GPCR) signaling and trafficking, and their knockdown typically leads to a decrease in agonist-induced ERK1/2 MAP kinase activation. Interestingly, for some GPCRs, knockdown of ßarr1 augments agonist-induced ERK1/2 phosphorylation although a mechanistic basis for this intriguing phenomenon is unclear. Here, we use selected GPCRs to explore a possible correlation between the spatial positioning of receptor phosphorylation sites and the contribution of ßarr1 in ERK1/2 activation. We discover that engineering a spatially positioned double-phosphorylation-site cluster in the bradykinin receptor (B2 R), analogous to that present in the vasopressin receptor (V2 R), reverses the contribution of ßarr1 in ERK1/2 activation from inhibitory to promotive. An intrabody sensor suggests a conformational mechanism for this role reversal of ßarr1, and molecular dynamics simulation reveals a bifurcated salt bridge between this double-phosphorylation site cluster and Lys294 in the lariat loop of ßarr1, which directs the orientation of the lariat loop. Our findings provide novel insights into the opposite roles of ßarr1 in ERK1/2 activation for different GPCRs with a direct relevance to biased agonism and novel therapeutics.
Subject(s)
MAP Kinase Signaling System , Receptors, G-Protein-Coupled , HEK293 Cells , Humans , Phosphorylation , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , beta-Arrestin 1/metabolism , beta-Arrestins/metabolismABSTRACT
The ß1-adrenoceptor (ß1AR) is a G-protein-coupled receptor (GPCR) that couples1 to the heterotrimeric G protein Gs. G-protein-mediated signalling is terminated by phosphorylation of the C terminus of the receptor by GPCR kinases (GRKs) and by coupling of ß-arrestin 1 (ßarr1, also known as arrestin 2), which displaces Gs and induces signalling through the MAP kinase pathway2. The ability of synthetic agonists to induce signalling preferentially through either G proteins or arrestins-known as biased agonism3-is important in drug development, because the therapeutic effect may arise from only one signalling cascade, whereas the other pathway may mediate undesirable side effects4. To understand the molecular basis for arrestin coupling, here we determined the cryo-electron microscopy structure of the ß1AR-ßarr1 complex in lipid nanodiscs bound to the biased agonist formoterol5, and the crystal structure of formoterol-bound ß1AR coupled to the G-protein-mimetic nanobody6 Nb80. ßarr1 couples to ß1AR in a manner distinct to that7 of Gs coupling to ß2AR-the finger loop of ßarr1 occupies a narrower cleft on the intracellular surface, and is closer to transmembrane helix H7 of the receptor when compared with the C-terminal α5 helix of Gs. The conformation of the finger loop in ßarr1 is different from that adopted by the finger loop of visual arrestin when it couples to rhodopsin8. ß1AR coupled to ßarr1 shows considerable differences in structure compared with ß1AR coupled to Nb80, including an inward movement of extracellular loop 3 and the cytoplasmic ends of H5 and H6. We observe weakened interactions between formoterol and two serine residues in H5 at the orthosteric binding site of ß1AR, and find that formoterol has a lower affinity for the ß1AR-ßarr1 complex than for the ß1AR-Gs complex. The structural differences between these complexes of ß1AR provide a foundation for the design of small molecules that could bias signalling in the ß-adrenoceptors.
Subject(s)
Cryoelectron Microscopy , Formoterol Fumarate/chemistry , Formoterol Fumarate/metabolism , Receptors, Adrenergic, beta-1/chemistry , Receptors, Adrenergic, beta-1/ultrastructure , beta-Arrestin 1/chemistry , beta-Arrestin 1/ultrastructure , Amino Acid Sequence , Animals , Binding Sites , GTP-Binding Protein alpha Subunits, Gs/chemistry , GTP-Binding Protein alpha Subunits, Gs/metabolism , GTP-Binding Protein alpha Subunits, Gs/ultrastructure , HEK293 Cells , Humans , Models, Molecular , Multiprotein Complexes , Receptors, Adrenergic, beta-1/metabolism , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/metabolism , Single-Chain Antibodies/ultrastructure , Zebrafish , beta-Arrestin 1/metabolismABSTRACT
ß-Arrestins (ßarrs) critically regulate G-protein-coupled receptor (GPCR) signaling and trafficking. ßarrs have two isoforms, ßarr1 and ßarr2. Receptor phosphorylation is a key determinant for the binding of ßarrs, and understanding the intricate details of receptor-ßarr interaction is the next frontier in GPCR structural biology. The high-resolution structure of active ßarr1 in complex with a phosphopeptide derived from GPCR has been revealed, but that of ßarr2 remains elusive. Here, we present a 2.3-Å crystal structure of ßarr2 in complex with a phosphopeptide (C7pp) derived from the carboxyl terminus of CXCR7. The structural analysis of C7pp-bound ßarr2 reveals key differences from the previously determined active conformation of ßarr1. One of the key differences is that C7pp-bound ßarr2 shows a relatively small inter-domain rotation. Antibody-fragment-based conformational sensor and hydrogen/deuterium exchange experiments further corroborated the structural features of ßarr2 and suggested that ßarr2 adopts a range of inter-domain rotations.
Subject(s)
Receptors, CXCR/metabolism , beta-Arrestin 2/chemistry , Calorimetry , Crystallography, X-Ray , HEK293 Cells , Humans , Phosphopeptides/chemistry , Phosphopeptides/metabolism , Phosphorylation , Protein Domains , Protein Transport/drug effects , Receptors, CXCR/chemistry , beta-Arrestin 2/agonists , beta-Arrestin 2/genetics , beta-Arrestin 2/metabolismABSTRACT
Agonist stimulation of G-protein-coupled receptors (GPCRs) typically leads to phosphorylation of GPCRs and binding to multifunctional proteins called ß-arrestins (ßarrs). The GPCR-ßarr interaction critically contributes to GPCR desensitization, endocytosis, and downstream signaling, and GPCR-ßarr complex formation can be used as a generic readout of GPCR and ßarr activation. Although several methods are currently available to monitor GPCR-ßarr interactions, additional sensors to visualize them may expand the toolbox and complement existing methods. We have previously described antibody fragments (FABs) that recognize activated ßarr1 upon its interaction with the vasopressin V2 receptor C-terminal phosphopeptide (V2Rpp). Here, we demonstrate that these FABs efficiently report the formation of a GPCR-ßarr1 complex for a broad set of chimeric GPCRs harboring the V2R C terminus. We adapted these FABs to an intrabody format by converting them to single-chain variable fragments and used them to monitor the localization and trafficking of ßarr1 in live cells. We observed that upon agonist simulation of cells expressing chimeric GPCRs, these intrabodies first translocate to the cell surface, followed by trafficking into intracellular vesicles. The translocation pattern of intrabodies mirrored that of ßarr1, and the intrabodies co-localized with ßarr1 at the cell surface and in intracellular vesicles. Interestingly, we discovered that intrabody sensors can also report ßarr1 recruitment and trafficking for several unmodified GPCRs. Our characterization of intrabody sensors for ßarr1 recruitment and trafficking expands currently available approaches to visualize GPCR-ßarr1 binding, which may help decipher additional aspects of GPCR signaling and regulation.
Subject(s)
Receptors, G-Protein-Coupled/metabolism , beta-Arrestin 1/metabolism , HEK293 Cells , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/metabolism , Protein Transport , Receptors, G-Protein-Coupled/genetics , beta-Arrestin 1/geneticsABSTRACT
ß-arrestins (ßarrs) play multifaceted roles in the signaling and regulation of G-protein-coupled receptors (GPCRs) including their desensitization and endocytosis. Recently determined cryo-EM structures of two different GPCRs in complex with ßarr1 provide the first glimpse of GPCR-ßarr engagement and a structural framework to understand their interaction.
Subject(s)
Receptors, G-Protein-Coupled/ultrastructure , beta-Arrestins/metabolism , beta-Arrestins/ultrastructure , Arrestins/metabolism , Endocytosis/physiology , GTP-Binding Proteins/metabolism , Humans , Phosphorylation , Protein Binding , Protein Isoforms/ultrastructure , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/physiology , Structure-Activity Relationship , beta-Arrestin 1/metabolism , beta-Arrestin 2/metabolismABSTRACT
Intrauterine bony fragments (IUBF) are an unusual finding in hysterectomy specimen received in a histopathology laboratory. Females harboring IUBF may present non-specific symptoms like vaginal bleeding, leukorrhea, chronic pelvic pain, and secondary infertility. Herein we report the case of a 35-year-old female who presented vaginal discharge and bleeding for two years, since when she had an abortion. Later, hysterectomy specimen revealed bone pieces in the uterine cavity.
Subject(s)
Humans , Female , Adult , Hysterectomy/adverse effects , Abortion, Incomplete , Dilatation and Curettage , AbortionABSTRACT
Desensitization, signaling, and trafficking of G-protein-coupled receptors (GPCRs) are critically regulated by multifunctional adaptor proteins, ß-arrestins (ßarrs). The two isoforms of ßarrs (ßarr1 and 2) share a high degree of sequence and structural similarity; still, however, they often mediate distinct functional outcomes in the context of GPCR signaling and regulation. A mechanistic basis for such a functional divergence of ßarr isoforms is still lacking. By using a set of complementary approaches, including antibody-fragment-based conformational sensors, we discover structural differences between ßarr1 and 2 upon their interaction with activated and phosphorylated receptors. Interestingly, domain-swapped chimeras of ßarrs display robust complementation in functional assays, thereby linking the structural differences between receptor-bound ßarr1 and 2 with their divergent functional outcomes. Our findings reveal important insights into the ability of ßarr isoforms to drive distinct functional outcomes and underscore the importance of integrating this aspect in the current framework of biased agonism.
Subject(s)
beta-Arrestins/chemistry , HEK293 Cells , Humans , Molecular Dynamics Simulation , Protein Domains , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Sequence Homology, Amino Acid , Signal Transduction , beta-Arrestins/genetics , beta-Arrestins/metabolismABSTRACT
The human complement component, C5a, binds two different seven-transmembrane receptors termed C5aR1 and C5aR2. C5aR1 is a prototypical G-protein-coupled receptor that couples to the Gαi subfamily of heterotrimeric G-proteins and ß-arrestins (ßarrs) following C5a stimulation. Peptide fragments derived from the C terminus of C5a can still interact with the receptor, albeit with lower affinity, and can act as agonists or antagonists. However, whether such fragments might display ligand bias at C5aR1 remains unexplored. Here, we compare C5a and a modified C-terminal fragment of C5a, C5apep, in terms of G-protein coupling, ßarr recruitment, endocytosis, and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activation at the human C5aR1. We discover that C5apep acts as a full agonist for Gαi coupling as measured by cAMP response and extracellular signal-regulated kinase 1/2 phosphorylation, but it displays partial agonism for ßarr recruitment and receptor endocytosis. Interestingly, C5apep exhibits full-agonist efficacy with respect to inhibiting lipopolysaccharide-induced interleukin-6 secretion in human macrophages, but its ability to induce human neutrophil migration is substantially lower compared with C5a, although both these responses are sensitive to pertussis toxin treatment. Taken together, our data reveal that compared with C5a, C5apep exerts partial efficacy for ßarr recruitment, receptor trafficking, and neutrophil migration. Our findings therefore uncover functional bias at C5aR1 and also provide a framework that can potentially be extended to chemokine receptors, which also typically interact with chemokines through a biphasic mechanism.
Subject(s)
Complement C5a/metabolism , Endocytosis , Receptor, Anaphylatoxin C5a/metabolism , beta-Arrestins/metabolism , Amino Acid Sequence , Cell Movement , Complement C5a/genetics , HEK293 Cells , Humans , Interleukin-6/genetics , Interleukin-6/metabolism , Macrophages/cytology , Macrophages/metabolism , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Neutrophils/metabolism , Phosphorylation , Protein Binding , Receptor, Anaphylatoxin C5a/genetics , Sequence Homology , Signal Transduction , beta-Arrestins/geneticsABSTRACT
G protein-coupled receptors (GPCRs) recognize a diverse array of extracellular stimuli, and they mediate a broad repertoire of signaling events involved in human physiology. Although the major effort on targeting GPCRs has typically been focused on their extracellular surface, a series of recent developments now unfold the possibility of targeting them from the intracellular side as well. Allosteric modulators binding to the cytoplasmic surface of GPCRs have now been described, and their structural mechanisms are elucidated by high-resolution crystal structures. Furthermore, pepducins, aptamers, and intrabodies targeting the intracellular face of GPCRs have also been successfully utilized to modulate receptor signaling. Moreover, small molecule compounds, aptamers, and synthetic intrabodies targeting ß-arrestins have also been discovered to modulate GPCR endocytosis and signaling. Here, we discuss the emerging paradigm of intracellular targeting of GPCRs, and outline the current challenges, potential opportunities, and future outlook in this particular area of GPCR biology.
Subject(s)
Endocytosis , Models, Molecular , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Allosteric Regulation/drug effects , Animals , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Aptamers, Nucleotide/pharmacology , Binding Sites , Endocytosis/drug effects , Humans , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/metabolism , Immunoglobulin Fragments/pharmacology , Ligands , Lipopeptides/chemistry , Lipopeptides/metabolism , Lipopeptides/pharmacology , Protein Conformation , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/chemistry , Signal Transduction/drug effectsABSTRACT
A case of thymoma with extensive ossification in an 8-year-old female child is presented. The presence of extensive ossification in the stroma of the thymoma is an extremely rare feature. To date, there is a single report of ossifying thymoma in children. This report represents the second known case in a child in the worldwide literature.
Subject(s)
Calcinosis/pathology , Thymoma/pathology , Thymoma/surgery , Thymus Neoplasms/pathology , Thymus Neoplasms/surgery , Biopsy, Needle , Child , Female , Follow-Up Studies , Humans , Immunohistochemistry , Rare Diseases , Severity of Illness Index , Sternotomy/methods , Treatment OutcomeABSTRACT
CONTEXT: To identify various causes, risk factors, age and sex distribution associated with sudden and unexpected natural deaths (SUNDs) in young adults of age between 18 and 35 years. MATERIALS AND METHODS: Retrospective analysis of autopsy reports and medical records of all SUNDs that occurred instantaneously or within 24 hours of onset of symptoms in young adults, between 2001 and 2009. RESULT: Of the total 6453 deaths autopsied during 2001-2009, 64 (0.99%) were SUNDs in young adults, chiefly in males between 30 and 35 years of age. Non-cardiac causes significantly predominated (73.4%) over cardiac causes (7.8%). Most of the SUND cases were due to preventable causes, including infections (54.6% cases), cerebrovascular accidents (9.37%) and ischemic cardiac causes (6.25%). Sudden adult death syndrome (SADS) accounted for 18.75% deaths. CONCLUSION: SUND in young adults is preventable. A meticulous post-mortem examination with special attention to the conduction system of heart and detailed toxicological analysis can pinpoint the cause of death in SADS.