Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide
that plays a major role in sensory neurotransmission. The CGRP receptor is a
heterodimer of the calcitonin receptor-like ...receptor (CLR) class B
G-protein-coupled receptor and the type 1 transmembrane domain protein, receptor
activity modifying protein (RAMP) 1. Herein, we report the 3.3 Å
structure of the human CGRP receptor in complex with CGRP and the Gs-protein
heterotrimer determined by Volta phase plate cryo-electron microscopy. The RAMP
transmembrane domain sits at the interface between transmembrane domains 3, 4
and 5 of CLR, and stabilises CLR extracellular loop 2. RAMP1 makes only limited
direct interaction with CGRP, consistent with allosteric modulation of CLR as
its key function. Molecular dynamics simulations indicate that RAMP1 provides
stability to the receptor complex, particularly the location of the CLR
extracellular domain. The work provides novel insight into the control of
G-protein-coupled receptor function.
Despite recent advances in crystallography and the availability of G-protein-coupled receptor (GPCR) structures, little is known about the mechanism of their activation process, as only the β2 ...adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously bound to the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into the activation mechanism and allosteric modulation of muscarinic receptors.
Monocyte-like cell lines (MCLCs), including THP-1, HL-60 and U-937 cells, are used routinely as surrogates for isolated human peripheral blood mononuclear cells (PBMCs). To systematically evaluate ...these immortalised cells and PBMCs as model systems to study inflammation relevant to the pathogenesis of type II diabetes and immuno-metabolism, we compared mRNA expression of inflammation-relevant genes, cell surface expression of cluster of differentiation (CD) markers, and chemotactic responses to inflammatory stimuli. Messenger RNA expression analysis suggested most genes were present at similar levels across all undifferentiated cells, though notably, IDO1, which encodes for indoleamine 2,3-dioxygenase and catabolises tryptophan to kynureninase (shown to be elevated in serum from diabetic patients), was not expressed in any PMA-treated MCLC, but present in GM-CSF-treated PBMCs. There was little overall difference in the pattern of expression of CD markers across all cells, though absolute expression levels varied considerably and the correlation between MCLCs and PBMCs was improved upon MCLC differentiation. Functionally, THP-1 and PBMCs migrated in response to chemoattractants in a transwell assay, with varying sensitivity to MCP-1, MIP-1α and LTB-4. However, despite similar gene and CD expression profiles, U-937 cells were functionally impaired as no migration was observed to any chemoattractant. Our analysis reveals that the MCLCs examined only partly replicate the genotypic and phenotypic properties of human PBMCs. To overcome such issues a universal differentiation protocol should be implemented for these cell lines, similar to those already used with isolated monocytes. Although not perfect, in our hands the THP-1 cells represent the closest, simplified surrogate model of PBMCs for study of inflammatory cell migration.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Muscarinic M1-M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor ...subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer's disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.
The glucagon-like peptide (GLP)-1 receptor (GLP-1R) is a class B G protein-coupled receptor (GPCR) that mediates the action of GLP-1, a peptide hormone secreted from three major tissues in humans, ...enteroendocrine L cells in the distal intestine, α cells in the pancreas, and the central nervous system, which exerts important actions useful in the management of type 2 diabetes mellitus and obesity, including glucose homeostasis and regulation of gastric motility and food intake. Peptidic analogs of GLP-1 have been successfully developed with enhanced bioavailability and pharmacological activity. Physiologic and biochemical studies with truncated, chimeric, and mutated peptides and GLP-1R variants, together with ligand-bound crystal structures of the extracellular domain and the first three-dimensional structures of the 7-helical transmembrane domain of class B GPCRs, have provided the basis for a two-domain-binding mechanism of GLP-1 with its cognate receptor. Although efforts in discovering therapeutically viable nonpeptidic GLP-1R agonists have been hampered, small-molecule modulators offer complementary chemical tools to peptide analogs to investigate ligand-directed biased cellular signaling of GLP-1R. The integrated pharmacological and structural information of different GLP-1 analogs and homologous receptors give new insights into the molecular determinants of GLP-1R ligand selectivity and functional activity, thereby providing novel opportunities in the design and development of more efficacious agents to treat metabolic disorders.
Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the ...molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.
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•Effect of mutation on affinity and efficacy of biased ligands mapped onto 3D models•Biased agonists form distinct interactions with the GLP-1R extracellular surface•Engagement of unique elements of the extracellular surface promotes biased agonism•Insights into class B GPCR activation/biased agonism can aid rational drug design
Understanding how different ligands engage and activate signaling pathways downstream of the GLP-1 receptor, a key type II diabetes target, suggests strategies for shaping signaling outcomes to favor physiologically beneficial effects.
Although classical approaches to G-protein-coupled receptor (GPCR) drug design have targeted the orthosteric binding site, potentially all GPCRs possess druggable allosteric sites. In addition, it is ...clear that GPCRs can adopt multiple active states linked to distinct functional outcomes that can be stabilized by both allosteric and orthosteric ligands. Recent studies have begun to explore the possibilities of linking orthosteric and allosteric pharmacophores to yield ‘bitopic’ ligands as an approach to achieve improved receptor affinity or selectivity. Furthermore, it is possible that previously identified functionally selective drugs may represent unappreciated bitopic ligands at this important class of drug targets. Here we discuss both the potential of bitopic ligands in GPCR drug discovery and the challenges associated with the design of such ligands.
Recently, the concept of ligand-directed signaling—the ability of different ligands of an individual receptor to promote distinct patterns of cellular response—has gained much traction in the field ...of drug discovery, with the potential to sculpt biological response to favor therapeutically beneficial signaling pathways over those leading to harmful effects. However, there is limited understanding of the mechanistic basis underlying biased signaling. The glucagon-like peptide-1 receptor is a major target for treatment of type-2 diabetes and is subject to ligand-directed signaling. Here, we demonstrate the importance of polar transmembrane residues conserved within family B G protein-coupled receptors, not only for protein folding and expression, but also in controlling activation transition, ligand-biased, and pathway-biased signaling. Distinct clusters of polar residues were important for receptor activation and signal preference, globally changing the profile of receptor response to distinct peptide ligands, including endogenous ligands glucagon-like peptide-1, oxyntomodulin, and the clinically used mimetic exendin-4.
G protein–coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic ...coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here, we used cryo-electron microscopy (cryo-EM) to determine structures of the cholecystokinin (CCK) type 1 receptor (CCK1R) bound to the CCK peptide agonist, CCK-8 and 2 distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11–GPCR complexes, the Gq–α5 helix (αH5) bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket were essentially equivalent when Gs was bound, with the Gs αH5 displaying a conformation that arises from “unwinding” of the far carboxyl-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein are likely to play critical roles in the promiscuous coupling of individual GPCRs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Drugs that allosterically target the human calcium-sensing receptor （CaSR） have substantial therapeutic potential, but are currently limited. Given the absence of high-resolution structures of the ...CaSR, we combined mutagenesis with a novel analytical approach and molecular modeling to develop an ＂enriched＂ picture of structure-function requirements for interaction between CaZ＋o and allosteric modulators within the CaSR＇s 7 transmembrane （7TM） domain. An extended cavity that accommodates multiple binding sites for structurally diverse ligands was identified. Phenylalkylamines bind to a site that overlaps with a putative Ca2＋o-binding site and extends towards an extracellu- lar vestibule. In contrast, the structurally and pharmacologically distinct AC-265347 binds deeper within the 7TM domains. Furthermore, distinct amino acid networks were found to mediate cooperativity by different modulators. These findings may facilitate the rational design of aliosteric modulators with distinct and potentially pathway-biased pharmacological effects.
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