Regulation of mTORC1 by Upstream Stimuli

The mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth, metabolism, and autophagy. Extensive research has focused on pathways that activate mTORC1 like growth factors and amino acids; however, much less is known about signaling cues that directly inhibit mTORC1 activity. Here, we report that G-protein coupled receptors (GPCRs) paired to Gαs proteins increase cyclic adenosine 3’5’ monophosphate (cAMP) to activate protein kinase A (PKA) and inhibit mTORC1. Mechanistically, PKA phosphorylates the mTORC1 component Raptor on Ser 791, leading to decreased mTORC1 activity. Consistently, in cells where Raptor Ser 791 is mutated to Ala, mTORC1 activity is partially rescued even after PKA activation. Gαs-coupled GPCRs stimulation leads to inhibition of mTORC1 in multiple cell lines and mouse tissues. Our results uncover a signaling pathway that directly inhibits mTORC1, and suggest that GPCRs paired to Gαs proteins may be potential therapeutic targets for human diseases with hyperactivated mTORC1.

Download Full-text

Spatial Organization of Rho GTPase signaling by RhoGEF/RhoGAP proteins

10.1101/354316 ◽

2018 ◽

Cited By ~ 3

Author(s):

Paul M. Müller ◽

Juliane Rademacher ◽

Richard D. Bagshaw ◽

Keziban M. Alp ◽

Girolamo Giudice ◽

...

Keyword(s):

Rho Gtpases ◽

Spatial Organization ◽

Rho Gtpase ◽

Critical Role ◽

Control Cell ◽

Positional Information ◽

Small Gtpases ◽

G Protein Coupled Receptors ◽

Signaling Networks ◽

G Protein Coupled

AbstractRho GTPases control cell morphogenesis and thus fundamental processes in all eukaryotes. They are regulated by 145 RhoGEF and RhoGAP multi-domain proteins in humans. How the Rho signaling system is organized to generate localized responses in cells and prevent their spreading is not understood. Here, we systematically characterized the substrate specificities, localization and interactome of the RhoGEFs/RhoGAPs and revealed their critical role in contextualizing and spatially delimiting Rho signaling. They localize to multiple compartments providing positional information, are extensively interconnected to jointly coordinate their signaling networks and are widely autoinhibited to remain sensitive to local activation. RhoGAPs exhibit lower substrate specificity than RhoGEFs and may contribute to preserving Rho activity gradients. Our approach led us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling a Cdc42/RhoA crosstalk. The spatial organization of Rho signaling thus differs from other small GTPases and expands the repertoire of mechanisms governing localized signaling activity.

Download Full-text

Glutamine and asparagine activate mTORC1 independently of Rag GTPases

Journal of Biological Chemistry ◽

10.1074/jbc.ac119.011578 ◽

2020 ◽

Vol 295 (10) ◽

pp. 2890-2899 ◽

Cited By ~ 10

Author(s):

Delong Meng ◽

Qianmei Yang ◽

Huanyu Wang ◽

Chase H. Melick ◽

Rishika Navlani ◽

...

Keyword(s):

Amino Acids ◽

Control Cell ◽

Nutrient Sensing ◽

Rag Gtpases ◽

Independent Mechanism ◽

Growth Metabolism ◽

Rag Gtpase ◽

Mtorc1 Activation ◽

Mtor Complex 1 ◽

Adp Ribosylation Factor

Nutrient sensing by cells is crucial, and when this sensing mechanism is disturbed, human disease can occur. mTOR complex 1 (mTORC1) senses amino acids to control cell growth, metabolism, and autophagy. Leucine, arginine, and methionine signal to mTORC1 through the well-characterized Rag GTPase signaling pathway. In contrast, glutamine activates mTORC1 through a Rag GTPase–independent mechanism that requires ADP-ribosylation factor 1 (Arf1). Here, using several biochemical and genetic approaches, we show that eight amino acids filter through the Rag GTPase pathway. Like glutamine, asparagine signals to mTORC1 through Arf1 in the absence of the Rag GTPases. Both the Rag-dependent and Rag-independent pathways required the lysosome and lysosomal function for mTORC1 activation. Our results show that mTORC1 is differentially regulated by amino acids through two distinct pathways.

Download Full-text

Prospects for use of peptides and their derivatives, structurally corresponding to the G protein-coupled receptors, in medicine

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20156101019 ◽

2015 ◽

Vol 61 (1) ◽

pp. 19-29 ◽

Cited By ~ 1

Author(s):

A.O. Shpakov ◽

E.A. Shpakova

Keyword(s):

G Protein ◽

Intracellular Signaling ◽

High Efficiency ◽

Clinical Medicine ◽

Inflammatory Diseases ◽

G Protein Coupled Receptors ◽

Signaling Cascades ◽

G Protein Coupled

The regulation of signaling pathways involved in the control of many physiological functions is carried out via the heterotrimeric G protein-coupled receptors (GPCR). The search of effective and selective regulators of GPCR and intracellular signaling cascades coupled with them is one of the important problems of modern fundamental and clinical medicine. Recently data suggest that synthetic peptides and their derivatives, structurally corresponding to the intracellular and transmembrane regions of GPCR, can interact with high efficiency and selectivity with homologous receptors and influence, thus, the functional activity of intracellular signaling cascades and fundamental cellular processes controlled by them. GPCR-peptides are active in both in vitro and in vivo. They regulate hematopoiesis, angiogenesis and cell proliferation, inhibit tumor growth and metastasis, and prevent the inflammatory diseases and septic shock. These data show greatest prospects in the development of the new generations of drugs based on GPCR-derived peptides, capable of regulating the important functions of the organism.

Download Full-text

Regulators of G protein Signaling (RGS) proteins (version 2020.4) in the IUPHAR/BPS Guide to Pharmacology Database

IUPHAR/BPS Guide to Pharmacology CITE ◽

10.2218/gtopdb/f891/2020.4 ◽

2020 ◽

Vol 2020 (4) ◽

Author(s):

Katelin E. Ahlers-Dannen ◽

Mohammed Alqinyah ◽

Christopher Bodle ◽

Josephine Bou Dagher ◽

Bandana Chakravarti ◽

...

Keyword(s):

G Protein ◽

G Protein Coupled Receptors ◽

Signaling Cascades ◽

G Protein Signaling ◽

Rgs Proteins ◽

Protein Signaling ◽

Heterotrimeric G Proteins ◽

Gtp Hydrolysis ◽

Rgs Domain ◽

G Protein Coupled

Regulator of G protein Signaling, or RGS, proteins serve an important regulatory role in signaling mediated by G protein-coupled receptors (GPCRs). They all share a common RGS domain that directly interacts with active, GTP-bound Gα subunits of heterotrimeric G proteins. RGS proteins stabilize the transition state for GTP hydrolysis on Gα and thus induce a conformational change in the Gα subunit that accelerates GTP hydrolysis, thereby effectively turning off signaling cascades mediated by GPCRs. This GTPase accelerating protein (GAP) activity is the canonical mechanism of action for RGS proteins, although many also possess additional functions and domains. RGS proteins are divided into four families, R4, R7, R12 and RZ based on sequence homology, domain structure as well as specificity towards Gα subunits. For reviews on RGS proteins and their potential as therapeutic targets, see e.g. [160, 377, 411, 415, 416, 512, 519, 312, 6].

Download Full-text

An arrestin-1 surface opposite of its interface with photoactivated rhodopsin engages with enolase-1

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013043 ◽

2020 ◽

Vol 295 (19) ◽

pp. 6498-6508

Author(s):

Connie Jaqueline Miranda ◽

Nicole Fernandez ◽

Nader Kamel ◽

Daniel Turner ◽

Del Benzenhafer ◽

...

Keyword(s):

Amino Acids ◽

Molecular Model ◽

Signaling Cascades ◽

Contact Sites ◽

Binding Partners ◽

Protein Partners ◽

Fluorescence Quench ◽

Selective Substitution ◽

Additional Protein ◽

G Protein Coupled

Arrestin-1 is the arrestin family member responsible for inactivation of the G protein–coupled receptor rhodopsin in photoreceptors. Arrestin-1 is also well-known to interact with additional protein partners and to affect other signaling cascades beyond phototransduction. In this study, we investigated one of these alternative arrestin-1 binding partners, the glycolysis enzyme enolase-1, to map the molecular contact sites between these two proteins and investigate how the binding of arrestin-1 affects the catalytic activity of enolase-1. Using fluorescence quench protection of strategically placed fluorophores on the arrestin-1 surface, we observed that arrestin-1 primarily engages enolase-1 along a surface that is opposite of the side of arrestin-1 that binds photoactivated rhodopsin. Using this information, we developed a molecular model of the arrestin-1–enolase-1 complex, which was validated by targeted substitutions of charge-pair interactions. Finally, we identified the likely source of arrestin's modulation of enolase-1 catalysis, showing that selective substitution of two amino acids in arrestin-1 can completely remove its effect on enolase-1 activity while still remaining bound to enolase-1. These findings open up opportunities for examining the functional effects of arrestin-1 on enolase-1 activity in photoreceptors and their surrounding cells.

Download Full-text

Minireview: Nutrient Sensing by G Protein-Coupled Receptors

Molecular Endocrinology ◽

10.1210/me.2013-1100 ◽

2013 ◽

Vol 27 (8) ◽

pp. 1188-1197 ◽

Cited By ~ 49

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.

Download Full-text

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

Journal of Biological Chemistry ◽

10.1074/jbc.m707355200 ◽

2007 ◽

Vol 283 (3) ◽

pp. 1525-1533 ◽

Cited By ~ 113

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.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj20040231 ◽

2004 ◽

Vol 380 (2) ◽

pp. 361-369 ◽

Cited By ~ 8

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.

Download Full-text

Regulators of G protein Signaling (RGS) proteins (version 2020.5) in the IUPHAR/BPS Guide to Pharmacology Database

IUPHAR/BPS Guide to Pharmacology CITE ◽

10.2218/gtopdb/f891/2020.5 ◽

2020 ◽

Vol 2020 (5) ◽

Author(s):

Katelin E. Ahlers-Dannen ◽

Mohammed Alqinyah ◽

Christopher Bodle ◽

Josephine Bou Dagher ◽

Bandana Chakravarti ◽

...

Keyword(s):

G Protein ◽

G Protein Coupled Receptors ◽

Signaling Cascades ◽

G Protein Signaling ◽

Rgs Proteins ◽

Protein Signaling ◽

Heterotrimeric G Proteins ◽

Gtp Hydrolysis ◽

Rgs Domain ◽

G Protein Coupled

Regulator of G protein Signaling, or RGS, proteins serve an important regulatory role in signaling mediated by G protein-coupled receptors (GPCRs). They all share a common RGS domain that directly interacts with active, GTP-bound Gα subunits of heterotrimeric G proteins. RGS proteins stabilize the transition state for GTP hydrolysis on Gα and thus induce a conformational change in the Gα subunit that accelerates GTP hydrolysis, thereby effectively turning off signaling cascades mediated by GPCRs. This GTPase accelerating protein (GAP) activity is the canonical mechanism of action for RGS proteins, although many also possess additional functions and domains. RGS proteins are divided into four families, R4, R7, R12 and RZ based on sequence homology, domain structure as well as specificity towards Gα subunits. For reviews on RGS proteins and their potential as therapeutic targets, see e.g. [183, 411, 446, 450, 451, 558, 566, 345, 9].

Download Full-text