▪ Abstract In the mid to late 1980s, studies were published that provided the first evidence for the existence of glutamate receptors that are not ligand-gated cation channels but are coupled to effector systems through GTP-binding proteins. Since those initial reports, tremendous progress has been made in characterizing these metabotropic glutamate receptors (mGluRs), including cloning and characterization of cDNA that encodes a family of eight mGluR subtypes, several of which have multiple splice variants. Also, tremendous progress has been made in developing new highly selective mGluR agonists and antagonists and toward determining the physiologic roles of the mGluRs in mammalian brain. These findings have exciting implications for drug development and suggest that the mGluRs provide a novel target for development of therepeutic agents that could have a significant impact on neuropharmacology.

Pharmacology and functions of metabotropic glutamate receptors.

Heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human β2-adrenergic receptor–T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein–coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the β2-adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand-binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family.

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High-Resolution Crystal Structure of an Engineered Human β2-Adrenergic G Protein–Coupled Receptor

Prostanoids are the cyclooxygenase metabolites of arachidonic acid and include prostaglandin (PG) D2, PGE2, PGF2α, PGI2, and thromboxne A2. They are synthesized and released upon cell stimulation and act on cells in the vicinity of their synthesis to exert their actions. Receptors mediating the actions of prostanoids were recently identified and cloned. They are G protein-coupled receptors with seven transmembrane domains. There are eight types and subtypes of prostanoid receptors that are encoded by different genes but as a whole constitute a subfamily in the superfamily of the rhodopsin-type receptors. Each of the receptors was expressed in cultured cells, and its ligand-binding properties and signal transduction pathways were characterized. Moreover, domains and amino acid residues conferring the specificities of ligand binding and signal transduction are being clarified. Information also is accumulating as to the distribution of these receptors in the body. It is also becoming clear for some types of ...

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Prostanoid Receptors: Structures, Properties, and Functions

G protein-coupled receptors (GPCRs) are seven transmembrane proteins that form the largest single family of integral membrane receptors. GPCRs transduce information provided by extracellular stimuli into intracellular second messengers via their coupling to heterotrimeric G proteins and the subsequent regulation of a diverse variety of effector systems. Agonist activation of GPCRs also initiates processes that are involved in the feedback desensitization of GPCR responsiveness, the internalization of GPCRs, and the coupling of GPCRs to heterotrimeric G protein-independent signal transduction pathways. GPCR desensitization occurs as a consequence of G protein uncoupling in response to phosphorylation by both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). GRK-mediated receptor phosphorylation promotes the binding of beta-arrestins, which not only uncouple receptors from heterotrimeric G proteins but also target many GPCRs for internalization in clathrin-coated vesicles. beta-Arrestin-dependent endocytosis of GPCRs involves the direct interaction of the carboxyl-terminal tail domain of beta-arrestins with both beta-adaptin and clathrin. The focus of this review is the current and evolving understanding of the contribution of GRKs, beta-arrestins, and endocytosis to GPCR-specific patterns of desensitization and resensitization. In addition to their role as GPCR-specific endocytic adaptor proteins, beta-arrestins also serve as molecular scaffolds that foster the formation of alternative, heterotrimeric G protein-independent signal transduction complexes. Similar to what is observed for GPCR desensitization and resensitization, beta-arrestin-dependent GPCR internalization is involved in the intracellular compartmentalization of these protein complexes.

Evolving Concepts in G Protein-Coupled Receptor Endocytosis: The Role in Receptor Desensitization and Signaling

Among membrane‐bound receptors, the G protein‐coupled receptors (GPCRs) are certainly the most diverse. They have been very successful during evolution, being capable of transducing messages as different as photons, organic odorants, nucleotides, nucleosides, peptides, lipids and proteins. Indirect studies, as well as two‐dimensional crystallization of rhodopsin, have led to a useful model of a common ‘central core’, composed of seven transmembrane helical domains, and its structural modifications during activation. There are at least six families of GPCRs showing no sequence similarity. They use an amazing number of different domains both to bind their ligands and to activate G proteins. The fine‐tuning of their coupling to G proteins is regulated by splicing, RNA editing and phosphorylation. Some GPCRs have been found to form either homo‐ or heterodimers with a structurally different GPCR, but also with membrane‐bound proteins having one transmembrane domain such as nina‐A, odr‐4 or RAMP, the latter being involved in their targeting, function and pharmacology. Finally, some GPCRs are unfaithful to G proteins and interact directly, via their C‐terminal domain, with proteins containing PDZ and Enabled/VASP homology (EVH)‐like domains.

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Molecular tinkering of G protein‐coupled receptors: an evolutionary success

A Peptide Derived from a β2-Adrenergic Receptor Transmembrane Domain Inhibits Both Receptor Dimerization and Activation

Exposure of beta 2-adrenergic receptors to agonists causes a rapid desensitization of the receptor-stimulated adenylyl cyclase, associated with an increased phosphorylation of the receptor. Agonist-promoted phosphorylation of the beta 2-adrenergic receptor (beta 2AR) by protein kinase A and the beta-adrenergic receptor kinase (beta ARK) is believed to promote a functional uncoupling of the receptor from the guanyl nucleotide regulatory protein Gs. More recently, palmitoylation of Cys341 of the receptor has also been proposed to play an important role in the coupling of the beta 2-adrenergic receptor to Gs. Here we report that substitution of the palmitoylated cysteine by a glycine (Gly341 beta 2 AR) using site directed mutagenesis leads to a receptor being highly phosphorylated and largely uncoupled from Gs. In Chinese hamster fibroblasts (CHW), stably transfected with the human receptor cDNAs, the basal phosphorylation level of Gly341 beta 2AR was found to be approximately 4 times that of the wild type receptor. This elevated phosphorylation level was accompanied by a depressed ability of the receptor to stimulate the adenylyl cyclase and to form a guanyl nucleotide-sensitive high affinity state for agonists. Moreover, exposure of this unpalmitoylated receptor to an agonist did not promote any further phosphorylation or uncoupling. A modest desensitization of the receptor-stimulated adenylyl cyclase response was observed but resulted from the agonist-induced sequestration of the unpalmitoylated receptor and could be blocked by concanavalin A. This contrasts with the agonist-promoted phosphorylation and uncoupling of the wild type receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1993.tb05663.x

Altered phosphorylation and desensitization patterns of a human beta 2-adrenergic receptor lacking the palmitoylated Cys341.

Palmitoylated Cysteine 341 Modulates Phosphorylation of the β2-Adrenergic Receptor by the cAMP-dependent Protein Kinase

Although palmitoylation of the β2-adrenergic receptor (β2AR), as well as its phosphorylation by the cyclic AMP-dependant protein kinase (PKA) and the β-adrenergic receptor kinase (βARK), are known to play important roles in agonist-promoted desensitization, their relative contribution and mutual regulatory influences are still poorly understood. In this study, we investigated the role that the carboxyl tail PKA site (Ser345,346) of the β2AR plays in its rapid agonist-promoted phosphorylation and desensitization. Mutation of this site (Ala345,346β2AR) significantly reduced the rate and extent of the rapid desensitization promoted by sustained treatment with the agonist isoproterenol. The direct contribution of Ser345,346 in desensitization was then studied by mutating all other putative PKA and βARK phosphorylation sites (Ala261,262βARK−β2AR). We found this mutant receptor to be phosphorylated upon receptor activation but not following direct activation of PKA, suggesting a role in receptor-specific (homologous) but not heterologous phosphorylation. However, despite its phosphorylated state, Ala261,262βARK−β2AR did not undergo rapid desensitization upon agonist treatment, indicating that phosphorylation of Ser345,346 alone is not sufficient to promote desensitization. Taken with the observation that mutation of either Ser345,346 or of the βARK phosphorylation sites prevented both the hyper-phosphorylation and constitutive desensitization of a palmitoylation-less mutant (Gly341β2AR), our data suggest a concerted/synergistic action of the two kinases that depends on the palmitoylation state of the receptor. Consistent with this notion, in vitro phosphorylation of Gly341β2AR by the catalytic subunit of PKA facilitated further phosphorylation of the receptor by purified βARK. Our study therefore allows us to propose a coordinated mechanism by which sequential depalmitoylation, and phosphorylation by PKA and βARK lead to the functional uncoupling and desensitization of the β2AR.

The palmitoylation state of the β2-adrenergic receptor regulates the synergistic action of cyclic AMP-dependent protein kinase and β-adrenergic receptor kinase involved in its phosphorylation and desensitization

Introduction In recent years, covalent modification of proteins by lipi'ds has been found to be a more frequent modification than originally anticipated [ 11. Three major classes of such modifications have been particularly well characterized. These are: (a) the N-terminal myristoylation of glycine residues through amide linkage; (b) the prenylation of cysteine residues, located in C-terminal domains, via the formation of thioether links to farnesyl or geranylgeranyl moieties; and (c) the palmitoylation which occurs through the thioesterification of cysteine residues, located in various domains of proteins, with palmitic acid. In contrast to myristylation and prenylation, which typically occur cotranslationally, palmitoylation is a genuine post-translational modification which can undergo dynamic regulation and which, in some cases, can be modulated by external stimuli [2-41. This modification has been found to be particularly prevalent for proteins involved in processes such as cell adhesion, cell growth and signal transduction, raising the intriguing possibility that it could play regulatory roles in these processes. Palmitoylated proteins include p2 1 rds [ 51, GAP-43 [ 61, several tyrosine kinases belonging to the p60"" family such as p56"" [7], p59'Y", pSS'gr and ~ 5 6 ' " ~ [8], as well as many G-protein a-subunits [9]. For these proteins, the attachment of the 16-carbon-long fatty acid has been proposed to be essential for their targeting to the inner face of the plasma membrane, the site of their biological functions [ 10-131. However, the observation that integral membrane proteins, which

Serge Moffett

Papers

A Peptide Derived from a β2-Adrenergic Receptor Transmembrane Domain Inhibits Both Receptor Dimerization and Activation

Altered phosphorylation and desensitization patterns of a human beta 2-adrenergic receptor lacking the palmitoylated Cys341.

Palmitoylated Cysteine 341 Modulates Phosphorylation of the β2-Adrenergic Receptor by the cAMP-dependent Protein Kinase

The palmitoylation state of the β2-adrenergic receptor regulates the synergistic action of cyclic AMP-dependent protein kinase and β-adrenergic receptor kinase involved in its phosphorylation and desensitization

Palmitoylation of G-protein-coupled receptors: a dynamic modification with functional consequences.