Non-canonical signaling and localizations of heterotrimeric G proteins

Hewavitharana, Thamara; Wedegaertner, Philip

doi:10.1016/j.cellsig.2011.08.014

Cited by 93 publications

(69 citation statements)

References 111 publications

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“…G␣ s subunits are anchored to the plasma membrane in part by palmitoylation of a single N-terminal cysteine residue (18,19), and activation dramatically enhances the rate of palmitate turnover (20 -22). A cytosolic thioesterase, acyl protein thioesterase 1 (APT1), 2 has been shown to catalyze deacylation of G␣ s in vitro, and this activity is inhibited by association with G␤␥ (23). These findings suggest that activation may promote APT1-mediated net removal of palmitate and thereby release active G␣ s from the plasma membrane, but direct evidence supporting this model has been not been reported.…”

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confidence: 53%

“…However, it is becoming increasingly apparent that these signaling molecules have important functions in other cellular locations (2). Therefore, it is important to know how G proteins are distributed among various intracellular compartments, and how these proteins traffic between the plasma membrane, the cytosol, cytoskeletal elements, and intracellular organelles both in their resting state and when they are activated.…”

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confidence: 99%

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Activated G Protein Gαs Samples Multiple Endomembrane Compartments

Martin

Lambert

2016

Journal of Biological Chemistry

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Heterotrimeric G proteins are localized to the plasma membrane where they transduce extracellular signals to intracellular effectors. G proteins also act at intracellular locations, and can translocate between cellular compartments. For example, G␣ s can leave the plasma membrane and move to the cell interior after activation. However, the mechanism of G␣ s translocation and its intracellular destination are not known. Here we use bioluminescence resonance energy transfer (BRET) to show that after activation, G␣ s rapidly associates with the endoplasmic reticulum, mitochondria, and endosomes, consistent with indiscriminate sampling of intracellular membranes from the cytosol rather than transport via a specific vesicular pathway. The primary source of G␣ s for endosomal compartments is constitutive endocytosis rather than activity-dependent internalization. Recycling of G␣ s to the plasma membrane is complete 25 min after stimulation is discontinued. We also show that an acylation-deacylation cycle is important for the steady-state localization of G␣ s at the plasma membrane, but our results do not support a role for deacylation in activity-dependent G␣ s internalization.Heterotrimeric G proteins are best known for their role in transducing signals from the extracellular environment across the plasma membrane (1). However, it is becoming increasingly apparent that these signaling molecules have important functions in other cellular locations (2). Therefore, it is important to know how G proteins are distributed among various intracellular compartments, and how these proteins traffic between the plasma membrane, the cytosol, cytoskeletal elements, and intracellular organelles both in their resting state and when they are activated.The most well studied example of activity-dependent G protein trafficking occurs in the retina, where intense illumination promotes the dissociation of transducin from rod outer segment disks and the subsequent translocation of both G␣ t and G␤␥ into the rod inner segment (3). Activity-dependent translocation of G␣ and G␤␥ subunits occurs in other cells as well (4, 5), most notably for G␣ s , the subunit responsible for stimulation of adenylate cyclase. Stimulation of a cognate receptor or inhibition of GTPase activity promotes activation and translocation of G␣ s from the plasma membrane to the cell interior (6 -8).One important yet poorly understood aspect of G␣ s internalization is the intracellular destination of this G protein. Cell fractionation studies have shown that a large amount of constitutively active G␣ s is found in the soluble fraction, suggesting that active G␣ s may simply become soluble in the cytosol (8, 9). However, only a small amount of G␣ s enters the soluble fraction after receptor-mediated activation, consistent with vesicle-mediated internalization and continued association with membranes. Imaging studies have shown that internalized G␣ s can appear to be diffuse in the cytosol (8, 9), but association with intracellular vesicles has also been observed (10 -12)...

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confidence: 53%

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confidence: 99%

Activated G Protein Gαs Samples Multiple Endomembrane Compartments

Martin

Lambert

2016

Journal of Biological Chemistry

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“…Finally, the receptor variant might have an ability to signal from intracellular compartments as observed for other GPCRs (35,36). However, further studies are warranted to delineate the mechanism of the intracellular retention and/or function of the most common H0 haplotype.…”

Section: Discussionmentioning

confidence: 99%

Genetic Variations in the Human G Protein-coupled Receptor Class C, Group 6, Member A (GPRC6A) Control Cell Surface Expression and Function

Jørgensen

Have

Underwood

et al. 2017

Journal of Biological Chemistry

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Edited by Henrik G. DohlmanGPRC6A is a G protein-coupled receptor activated by Lamino acids, which, based on analyses of knock-out mice, has been suggested to have physiological functions in metabolism and testicular function. The human ortholog is, however, mostly retained intracellularly in contrast to the cell surface-expressed murine and goldfish orthologs. The latter orthologs are G q -coupled and lead to intracellular accumulation of inositol phosphates and calcium release. In the present study we cloned the bonobo chimpanzee GPRC6A receptor, which is 99% identical to the human receptor, and show that it is cell surface-expressed and functional. By analyses of chimeric human/mouse and human/bonobo receptors, bonobo receptor mutants, and the single nucleotide polymorphism database at NCBI, we identify an insertion/deletion variation in the third intracellular loop responsible for the intracellular retention and lack of function of the human ortholog. Genetic analyses of the 1000 genome database and the Inter99 cohort of 6,000 Danes establish the distribution of genotypes among ethnic groups, showing that the cell surface-expressed and functional variant is much more prevalent in the African population than in European and Asian populations and that this variant is partly linked with a stop codon early in the receptor sequence (rs6907580, amino acid position 57). In conclusion, our data solve a more than decade-old question of why the cloned human GPRC6A receptor is not cell surface-expressed and functional and provide a genetic framework to study human phenotypic traits in large genome sequencing projects linked with physiological measurement and biomarkers.Communication between the exterior and interior of cells is essential for cellular survival. A pivotal class of proteins, specialized to carry out such signal transduction, is the G proteincoupled receptors (GPCRs), 2 a family that includes ϳ800 subtypes in humans. The GPCR class C, group 6, member A (GPRC6A) belongs to the non-olfactory GPCRs as part of the class C receptors. Human GPRC6A (h6A) was cloned from a human kidney cDNA library in 2004 (1). Cloning and deorphanization of the mouse (2, 3) and rat (4) GPRC6A orthologs rapidly followed. These studies, together with recent evidence (5) support the existence of GPRC6A as a dimer on the cell surface. However, surprisingly, h6A has been shown to be retained intracellularly and thus does not respond to agonists, which contrasts findings for the mouse, rat, and goldfish orthologs (2, 3, 6). To deorphanize h6A, we generated chimeras between the human and goldfish GPRC6A orthologs. We found that a fusion of the human large extracellular amino-terminal domain (ATD) to the 7-transmembrane (7TM) and C-terminal domains of the orthologous goldfish 5.24 receptor allowed efficient surface expression of the chimera and thereby a way to

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“…G-protein α-and βγ-subunits were classically thought to act only at the plasma membrane, but recent findings suggest important new roles for G-protein subunits at intracellular locations (45)(46)(47). Whereas rapid diffusion of second messengers generated by G-protein effectors at the plasma membrane enables fast signal communication to other regions of the cell, βγ translocation to intracellular regions could enable more specific communication between the plasma membrane and internal membranes.…”

Section: Discussionmentioning

confidence: 99%

G-protein signaling leverages subunit-dependent membrane affinity to differentially control βγ translocation to intracellular membranes

O’Neill¹,

Karunarathne²,

Kalyanaraman³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Activation of G-protein heterotrimers by receptors at the plasma membrane stimulates βγ-complex dissociation from the α-subunit and translocation to internal membranes. This intermembrane movement of lipid-modified proteins is a fundamental but poorly understood feature of cell signaling. The differential translocation of G-protein βγ-subunit types provides a valuable experimental model to examine the movement of signaling proteins between membranes in a living cell. We used live cell imaging, mathematical modeling, and in vitro measurements of lipidated fluorescent peptide dissociation from vesicles to determine the mechanistic basis of the intermembrane movement and identify the interactions responsible for differential translocation kinetics in this family of evolutionarily conserved proteins. We found that the reversible translocation is mediated by the limited affinity of the βγ-subunits for membranes. The differential kinetics of the βγ-subunit types are determined by variations among a set of basic and hydrophobic residues in the γ-subunit types. Gprotein signaling thus leverages the wide variation in membrane dissociation rates among different γ-subunit types to differentially control βγ-translocation kinetics in response to receptor activation. The conservation of primary structures of γ-subunits across mammalian species suggests that there can be evolutionary selection for primary structures that confer specific membrane-binding affinities and consequent rates of intermembrane movement.protein-membrane interaction | spatio-temporal dynamics | G protein-coupled receptors C ompared with the vast biochemical information available for signaling proteins from cell-disruptive techniques, much less is known about their spatiotemporal dynamics in the 3D space of an intact living cell. Many important signaling proteins are posttranslationally modified with lipid groups that facilitate their interactions with membranes. Live cell imaging has so far revealed important roles for constitutive cycles that maintain proper localization of lipidated proteins (1) and for dynamic protein redistribution throughout the cell in response to a signal (2), but the detailed nature of lipid-modified protein movement between membranes is generally not well understood.Heterotrimeric G proteins consist of lipid-modified α-and βγ-subunits (3). They are central transducers of the most important signaling pathways in mammals, yet little is known about their intracellular movement. Inactive heterotrimers consist of a GDP-bound α-subunit in complex with a stable βγ-dimer and localize to the plasma membrane (3). Extracellular signals activate transmembrane G-protein-coupled receptors (GPCRs) that catalyze nucleotide exchange on the G-protein α-subunit (3). The βγ-complex can then dissociate from the active α-subunit and translocate from the plasma membrane to intracellular membranes (4-6). βγ translocation has been observed for many different cell types and receptor types, suggesting that it is a common feature of G-protein signaling (4-...

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Non-canonical signaling and localizations of heterotrimeric G proteins

Cited by 93 publications

References 111 publications

Activated G Protein Gαs Samples Multiple Endomembrane Compartments

Activated G Protein Gαs Samples Multiple Endomembrane Compartments

Genetic Variations in the Human G Protein-coupled Receptor Class C, Group 6, Member A (GPRC6A) Control Cell Surface Expression and Function

G-protein signaling leverages subunit-dependent membrane affinity to differentially control βγ translocation to intracellular membranes

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