scholarly journals Identification of a stretch of six divergent amino acids on the alpha5 helix of Galpha16 as a major determinant of the promiscuity and efficiency of receptor coupling

2004 ◽  
Vol 380 (2) ◽  
pp. 361-369 ◽  
Author(s):  
Maurice K. C. HO ◽  
Jasmine H. P. CHAN ◽  
Cecilia S. S. WONG ◽  
Yung H. WONG
Keyword(s):  
Amino Acids ◽  
Inositol Phosphate ◽  
G Protein Coupled Receptors ◽  
The Other ◽  
Effective Coupling ◽  
Receptor Coupling ◽  
Receptor Recognition ◽  
Inositol Phosphate Accumulation ◽  
Coupling Specificity ◽  
G Protein Coupled

A broad repertory of G-protein-coupled receptors shows effective coupling with the haematopoietic G16 protein. In the present study, individual residues along the C-terminal α5 helix of Gα16 were examined for their contributions in defining receptor-coupling specificity. Residues that are relatively conserved within, but diverse between, the subfamilies of cloned Gα subunits were mutated into the corresponding Gαz residues. Six Gi-linked receptors with different coupling efficiencies to Gα16 were examined for their ability to utilize the various Gα16 mutants to mediate agonist-induced inositol phosphate accumulation and Ca2+ mobilization. Co-operative enhancements of receptor coupling were observed with chimaeras harbouring multiple mutations at Glu350, Lys357 and Leu364 of Gα16. Mutation of Leu364 into isoleucine appeared to be more efficient in enhancing receptor recognition compared with mutations at the other two sites. Mutation of a stretch of six consecutive residues (362–367) lying towards the end of the α5 helix was found to broaden significantly the receptor-coupling profile of Gα16, and the effect was mediated partly through interactions with the β2–β3 loop. These results suggested that a stretch of six distinctive residues at the α5 helix of Gα16 is particularly important, whereas other discrete residues spreading along the α5 helix function co-operatively for determining the specificity of receptor recognition.

Molecular dissection of G protein preference using Gsα chimeras reveals novel ligand signaling of GPCRs

2007 ◽  
Vol 293 (4) ◽  
pp. E1021-E1029 ◽  
Author(s):  
Shih-Han Hsu ◽  
Ching-Wei Luo
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Adenylyl Cyclases ◽  
Pharmaceutical Development ◽  
Receptor Coupling ◽  
Physiological Processes ◽  
A1 Receptor ◽  
Coupling Specificity ◽  
First Time ◽  
G Protein Coupled

Although only 16 genes have been identified in mammals, several Gα subunits can be simultaneously activated by G protein-coupled receptors (GPCRs) to modulate their complicated functions. Current GPCR assays are limited in the evaluation of selective Gα activation, thus not allowing a comprehensive pathway screening. Because adenylyl cyclases are directly activated by Gsα and the carboxyl termini of the various Gα proteins determine their receptor coupling specificity, we proposed a set of chimeric Gsα where the COOH-terminal five amino acids are replaced by those of other Gα proteins and used these to dissect the potential Gα linked to a given GPCR. Unlike Gqα, G12α, and Giα outputs, compounding the signals from several Gα members, the chimeric Gsα proteins provide a superior molecular approach that reflects the previously uncharacterized pathways of GPCRs under the same cAMP platform. This is, to our knowledge, the first time allowing verification of the whole spectrum of Gα coupling preference of adenosine A1 receptor, reported to couple to multiple G proteins and modulate many physiological processes. Furthermore, we were able to distinguish the uncharacterized pathways between the two neuromedin U receptors (NMURs), which distribute differently but are stimulated by a common agonist. In contrast to the Gq signals mainly conducted by NMUR1, NMUR2 routed preferentially to the Gi pathways. Dissecting the potential Gα coupling to these GPCRs will promote an understanding of their physiological roles and benefit the pharmaceutical development of agonists/antagonists by exploiting the selective affinity toward a certain Gα subclass.

scholarly journals Minireview: Nutrient Sensing by G Protein-Coupled Receptors

2013 ◽  
Vol 27 (8) ◽  
pp. 1188-1197 ◽  
Author(s):  
Eric M. Wauson ◽  
Andrés Lorente-Rodríguez ◽  
Melanie H. Cobb
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
G Protein ◽  
Taste Receptor ◽  
Sweet Taste ◽  
Nutrient Sensing ◽  
G Protein Coupled Receptors ◽  
Amino Acid Availability ◽  
Class C ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) are membrane proteins that recognize molecules in the extracellular milieu and transmit signals inside cells to regulate their behaviors. Ligands for many GPCRs are hormones or neurotransmitters that direct coordinated, stereotyped adaptive responses. Ligands for other GPCRs provide information to cells about the extracellular environment. Such information facilitates context-specific decision making that may be cell autonomous. Among ligands that are important for cellular decisions are amino acids, required for continued protein synthesis, as metabolic starting materials and energy sources. Amino acids are detected by a number of class C GPCRs. One cluster of amino acid-sensing class C GPCRs includes umami and sweet taste receptors, GPRC6A, and the calcium-sensing receptor. We have recently found that the umami taste receptor heterodimer T1R1/T1R3 is a sensor of amino acid availability that regulates the activity of the mammalian target of rapamycin. This review focuses on an array of findings on sensing amino acids and sweet molecules outside of neurons by this cluster of class C GPCRs and some of the physiologic processes regulated by them.

Deciphering the specific role of Gαi/o isoforms: functional selective oxytocin ligands and somatostatin SST5 receptor mutants

2013 ◽  
Vol 41 (1) ◽  
pp. 166-171 ◽  
Author(s):  
Marta Busnelli ◽  
Erika Peverelli ◽  
Giovanna Mantovani ◽  
Anna Spada ◽  
Bice Chini
Keyword(s):  
Somatostatin Receptor ◽  
Receptor Activation ◽  
G Protein Coupled Receptors ◽  
Receptor Coupling ◽  
Biological Responses ◽  
Selective Ligands ◽  
Specific Effector ◽  
G Protein Coupled

Receptor coupling to different G-proteins and β-arrestins has been described for a number of GPCRs (G-protein-coupled receptors), suggesting a multi-state model of receptor activation in which each receptor can assume a number of different active conformations, each capable of promoting the coupling to a specific effector. Consistently, functional-selective ligands and biased agonists have been described to be able to induce and/or stabilize only a subset of specific active conformations. Furthermore, GPCR mutants deficient in selective coupling have been reported. Functional selective ligands and receptor mutants thus constitute unique tools to dissect the specific roles of different effectors, in particular among the Gi/o family. In the present mini-review, we focus on (i) the identification of functional selective OXT (oxytocin)-derived peptides capable of activating single Gi/o isoforms, namely Gi1 or Gi3; and (ii) the characterization of an SS (somatostatin) receptor SST5 mutant selectively impaired in its GoA coupling. These analogues and receptor mutants represent unique tools for examining the contribution of Gi/o isoforms in complex biological responses and open the way for the development of drugs with peculiar selectivity profiles.

scholarly journals Site-specific Incorporation of Keto Amino Acids into Functional G Protein-coupled Receptors Using Unnatural Amino Acid Mutagenesis

2007 ◽  
Vol 283 (3) ◽  
pp. 1525-1533 ◽  
Author(s):  
Shixin Ye ◽  
Caroline Köhrer ◽  
Thomas Huber ◽  
Manija Kazmi ◽  
Pallavi Sachdev ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
G Protein ◽  
Unnatural Amino Acids ◽  
Unnatural Amino Acid ◽  
G Protein Coupled Receptors ◽  
Calcium Flux ◽  
Unnatural Amino Acid Mutagenesis ◽  
Amino Acid Mutagenesis ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-l-phenylalanine (Acp) and p-benzoyl-l-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to ∼50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5–2 μg/107 cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors.

scholarly journals G protein signaling governing cell fate decisions involves opposing Gα subunits inCryptococcus neoformans

2007 ◽  
Vol 18 (9) ◽  
pp. 3237-3249 ◽  
Author(s):  
Yen-Ping Hsueh ◽  
Chaoyang Xue ◽  
Joseph Heitman
Keyword(s):  
G Protein ◽  
Cell Fate ◽  
G Protein Coupled Receptors ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Pheromone Receptor ◽  
Cell Fate Decisions ◽  
Regulatory Circuit ◽  
Receptor Recognition ◽  
G Protein Coupled

Communication between cells and their environments is often mediated by G protein-coupled receptors and cognate G proteins. In fungi, one such signaling cascade is the mating pathway triggered by pheromone/pheromone receptor recognition. Unlike Saccharomyces cerevisiae, which expresses two Gα subunits, most filamentous ascomycetes and basidiomycetes have three Gα subunits. Previous studies have defined the Gα subunit acting upstream of the cAMP-protein kinase A pathway, but it has been unclear which Gα subunit is coupled to the pheromone receptor and response pathway. Here we report that in the pathogenic basidiomycetous yeast Cryptococcus neoformans, two Gα subunits (Gpa2, Gpa3) sense pheromone and govern mating. gpa2 gpa3 double mutants, but neither gpa2 nor gpa3 single mutants, are sterile in bilateral crosses. By contrast, deletion of GPA3 (but not GPA2) constitutively activates pheromone response and filamentation. Expression of GPA2 and GPA3 is differentially regulated: GPA3 expression is induced by nutrient-limitation, whereas GPA2 is induced during mating. Based on the phenotype of dominant active alleles, Gpa2 and Gpa3 signal in opposition: Gpa2 promotes mating, whereas Gpa3 inhibits. The incorporation of an additional Gα into the regulatory circuit enabled increased signaling complexity and facilitated cell fate decisions involving choice between yeast growth and filamentous asexual/sexual development.

scholarly journals Site-Specific Fluorescent Labeling of Purified G-Protein-Coupled Receptors Using Genetically-Encoded Unnatural Amino Acids

2010 ◽  
Vol 98 (3) ◽  
pp. 291a-292a
Author(s):  
Shixin Ye ◽  
Manija A. Kazmi ◽  
Terence Duarte ◽  
Saranga Naganathan ◽  
Thomas P. Sakmar ◽  
...  
Keyword(s):  
Amino Acids ◽  
G Protein ◽  
Unnatural Amino Acids ◽  
Fluorescent Labeling ◽  
G Protein Coupled Receptors ◽  
Site Specific ◽  
G Protein Coupled

scholarly journals Multiplex Detection of Functional G Protein-Coupled Receptors Harboring Site-Specifically Modified Unnatural Amino Acids

Biochemistry ◽  
2015 ◽  
Vol 54 (3) ◽  
pp. 776-786 ◽  
Author(s):  
Saranga Naganathan ◽  
Sarmistha Ray-Saha ◽  
Minyoung Park ◽  
He Tian ◽  
Thomas P. Sakmar ◽  
...  
Keyword(s):  
Amino Acids ◽  
G Protein ◽  
Unnatural Amino Acids ◽  
G Protein Coupled Receptors ◽  
Multiplex Detection ◽  
G Protein Coupled

scholarly journals Regulation of mTORC1 by Upstream Stimuli

Genes ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 989
Author(s):  
Chase H. Melick ◽  
Jenna L. Jewell
Keyword(s):  
Amino Acids ◽  
Growth Factors ◽  
Control Cell ◽  
Mammalian Target Of Rapamycin ◽  
Small Gtpases ◽  
G Protein Coupled Receptors ◽  
Signaling Cascades ◽  
Growth Metabolism ◽  
Mtor Complex 1 ◽  
G Protein Coupled

The mammalian target of rapamycin (mTOR) is an evolutionary conserved Ser/Thr protein kinase that senses multiple upstream stimuli to control cell growth, metabolism, and autophagy. mTOR is the catalytic subunit of mTOR complex 1 (mTORC1). A significant amount of research has uncovered the signaling pathways regulated by mTORC1, and the involvement of these signaling cascades in human diseases like cancer, diabetes, and ageing. Here, we review advances in mTORC1 regulation by upstream stimuli. We specifically focus on how growth factors, amino acids, G-protein coupled receptors (GPCRs), phosphorylation, and small GTPases regulate mTORC1 activity and signaling.

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