Visualization of G Protein-coupled Receptor Trafficking with the Aid of the Green Fluorescent Protein

Tarasova, Nadya I.; Stauber, Roland H.; Choi, Joon Ki; Hudson, Eric A.; Czerwinski, Grzegorz; Miller, Jeffrey L.; Pavlakis, George N.; Michejda, Christopher J.; Wank, Stephen A.

doi:10.1074/jbc.272.23.14817

Cited by 167 publications

(158 citation statements)

References 42 publications

(32 reference statements)

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“…In contrast, GCR1-GFP fluorescence appeared in a punctuate pattern, implying an association with particular membrane structures or possible internalization (Fig. 5B), a pattern also found with some mammalian GPCR-GFP fusions (Chun et al, 1994;Tarasova et al, 1997;Kallal et al, 1998).…”

Section: Gcr1 Subcellular and Tissue Localizationsupporting

confidence: 56%

GCR1 Can Act Independently of Heterotrimeric G-Protein in Response to Brassinosteroids and Gibberellins in Arabidopsis Seed Germination

et al. 2004

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Signal recognition by seven-transmembrane (7TM) cell-surface receptors is typically coupled by heterotrimeric G-proteins to downstream effectors in metazoan, fungal, and amoeboid cells. Some responses perceived by 7TM receptors in amoeboid cells and possibly in human cells can initiate downstream action independently of heterotrimeric G-proteins. Plants use heterotrimeric G-protein signaling in the regulation of growth and development, particularly in hormonal control of seed germination, but it is not yet clear which of these responses utilize a 7TM receptor. Arabidopsis GCR1 has a predicted 7TM-spanning domain and other features characteristic of 7TM receptors. Loss-of-function gcr1 mutants indicate that GCR1 plays a positive role in gibberellin-(GA) and brassinosteroid-(BR) regulated seed germination. The null mutants of GCR1 are less sensitive to GA and BR in seed germination. This phenotype is similar to that previously observed for transcript null mutants in the Ga-subunit, gpa1. However, the reduced sensitivities toward GA and BR in the single gcr1, gpa1, and agb1 (heterotrimeric G-protein b-subunit) mutants are additive or synergistic in the double and triple mutants. Thus, GCR1, unlike a typical 7TM receptor, apparently acts independently of the heterotrimeric G-protein in at least some aspects of seed germination, suggesting that this alternative mode of 7TM receptor action also functions in the plant kingdom.Signaling through heterotrimeric G-proteins is highly conserved among divergent eukaryotes. G-proteins physically couple the recognition of many extracellular signals by cell-surface receptors to activation of enzyme activities in the cytoplasm. In the classical paradigm, ligand binding to its cognate GPCR activates receptor-mediated GDP/GTP exchange on the a-subunit (Ga), causing dissociation of Ga from the bg dimer (Gbg). Activated Ga-subunits, Gbg, or both then bind to downstream target proteins, which results in the relevant cellular responses (Gilman, 1987). There are 23 Ga-, 6 Gb-, and 12 Gg-subunits in humans (Vanderbeld and Kelly, 2000). In contrast to humans, the Arabidopsis genome contains genes encoding only one prototypical G-protein a-subunit (GPA1), one G-protein b-subunit (AGB1), and two G-protein g-subunits (AGG1 and AGG2), indicating that the repertoire of heterotrimeric G-protein complexes in plants is smaller (Assmann, 2002;Jones, 2002). Studies on the null alleles of GPA1 and AGB1 suggest that plants use heterotrimeric G-protein signaling in many growth and developmental processes (Ullah et al., , 2002(Ullah et al., , 2003Wang et al., 2001;Chen et al., 2003).No classical GPCR has been definitively identified in plants. To date, the most promising candidate for a plant GPCR remains GCR1, independently cloned by two groups (Josefsson and Rask, 1997; PlakidouDymock et al., 1998). GCR1 encodes a protein with predicted seven membrane-spanning domains and has some sequence similarity to Dictyostelium cAMP receptors. GCR1 was originally proposed to be a receptor for cytokinins (Plakid...

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Section: Gcr1 Subcellular and Tissue Localizationsupporting

confidence: 56%

GCR1 Can Act Independently of Heterotrimeric G-Protein in Response to Brassinosteroids and Gibberellins in Arabidopsis Seed Germination

et al. 2004

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“…Assessment of [Ca 2ϩ ] i -The measurements were carried out in Fura-2/AM-treated cells utilizing the Attofluor digital imaging system as described (15). Stock solution of antagonists (5-10 mM) in dimethyl sulfoxide were diluted in phosphate-buffered saline and added to the cells preloaded with Fura-2/AM.…”

Section: Methodsmentioning

confidence: 99%

“…CHO cells expressing CCKAR were obtained as described (15). Confocal laserscanning microscopy was performed as described (15).…”

Section: Methodsmentioning

confidence: 99%

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Inhibition of G-protein-coupled Receptor Function by Disruption of Transmembrane Domain Interactions

Tarasova¹,

Rice²,

Michejda³

1999

Journal of Biological Chemistry

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“…Green fluorescent protein (GFP) [21,22] from the jellyfish Aequoria ictoria has revolutionized cell biology, being widely used to ' tag ' proteins of interest and to study the dynamics of their subcellular trafficking in living cells in real time [21]. Many membrane-membrane associated proteins have been studied using this method, including TGN38 [23], G-protein-coupled receptors [24,25] and proteins which utilize the secretory pathway [26], allowing detailed analysis of the dynamics of the behaviour of these proteins in a variety of different experimental circumstances. To that end, we have reported the use of GFPtagged Glut4 expressed both in CHO cells and 3T3-L1 adipocytes [27,28].…”

Section: Introductionmentioning

confidence: 99%

Trafficking of Glut4–Green Fluorescent Protein chimaeras in 3T3-L1 adipocytes suggests distinct internalization mechanisms regulating cell surface Glut4 levels

Campbell

Tavaré

Leader

et al. 1999

Biochemical Journal

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Insulin stimulates glucose transport in adipose and muscle tissue by stimulating the movement (‘translocation’) of an intracellular pool of glucose transporters (the Glut4 isoform) to the plasma membrane. We have engineered a series of chimaeras between Glut4 and green fluorescent protein (GFP) from Aequoria victoria and expressed these proteins in 3T3-L1 adipocytes by microinjection of plasmid cDNA. In the absence of insulin, GFP-Glut4 is localized intracellularly within a perinuclear compartment and multiple intracellular punctate structures. In response to insulin, chimaeric GFP-Glut4 species exhibit a profound redistribution to the cell surface with kinetics comparable with the endogenous protein. The intracellular localization of GFP-Glut4 overlaps partially with compartments labelled with Texas Red transferrin, but is largely distinct from intracellular structures identified using Lysotracker-Red®. K+-depletion resulted in the accumulation of GFP-Glut4 at the cell surface, but to an lesser extent than that observed in response to insulin. In contrast with native Glut4, removal of the insulin stimulus or treatment of insulin-stimulated cells with phosphatidylinositol 3′-kinase inhibitors did not result in re-internalization of the chimaeric GFP-Glut4 from the plasma membrane, suggesting that the recycling properties of this species differ from the native Glut4 molecule. We suggest that the recycling pathway utilized by GFP-Glut4 in the absence of insulin is distinct from that used to internalize GFP-Glut4 from the plasma membrane after withdrawal of the insulin stimulus, which may reflect distinct pathways for internalization of endogenous Glut4 in the presence or absence of insulin.

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Visualization of G Protein-coupled Receptor Trafficking with the Aid of the Green Fluorescent Protein

Cited by 167 publications

References 42 publications

GCR1 Can Act Independently of Heterotrimeric G-Protein in Response to Brassinosteroids and Gibberellins in Arabidopsis Seed Germination

GCR1 Can Act Independently of Heterotrimeric G-Protein in Response to Brassinosteroids and Gibberellins in Arabidopsis Seed Germination

Inhibition of G-protein-coupled Receptor Function by Disruption of Transmembrane Domain Interactions

Trafficking of Glut4–Green Fluorescent Protein chimaeras in 3T3-L1 adipocytes suggests distinct internalization mechanisms regulating cell surface Glut4 levels

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