scholarly journals A pH-Sensitive Fluor, CypHerTM 5, Used to Monitor Agonist-Induced G Protein-Coupled Receptor Internalization in Live Cells

BioTechniques ◽  
2002 ◽  
Vol 33 (5) ◽  
pp. 1152-1157 ◽  
Author(s):  
E.J. Adie ◽  
S. Kalinka ◽  
L. Smith ◽  
M.J. Francis ◽  
A. Marenghi ◽  
...  
Keyword(s):  
G Protein ◽  
Ph Sensitive ◽  
Receptor Internalization ◽  
Live Cells ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

scholarly journals G protein–coupled receptor/arrestin3 modulation of the endocytic machinery

2002 ◽  
Vol 156 (4) ◽  
pp. 665-676 ◽  
Author(s):  
Francesca Santini ◽  
Ibragim Gaidarov ◽  
James H. Keen
Keyword(s):  
G Protein ◽  
Fluorescent Protein ◽  
De Novo ◽  
Membrane Skeleton ◽  
Live Cells ◽  
G Protein Coupled Receptor ◽  
Cortical Actin ◽  
Endocytic Machinery ◽  
Green Fluorescent ◽  
G Protein Coupled

Nonvisual arrestins (arr) modulate G protein–coupled receptor (GPCR) desensitization and internalization and bind to both clathrin (CL) and AP-2 components of the endocytic coated pit (CP). This raises the possibility that endocytosis of some GPCRs may be a consequence of arr-induced de novo CP formation. To directly test this hypothesis, we examined the behavior of green fluorescent protein (GFP)-arr3 in live cells expressing β2-adrenergic receptors and fluorescent CL. After agonist stimulation, the diffuse GFP-arr3 signal rapidly became punctate and colocalized virtually completely with preexisting CP spots, demonstrating that activated complexes accumulate in previously formed CPs rather than nucleating new CP formation. After arr3 recruitment, CP appeared larger: electron microscopy analysis revealed an increase in both CP number and in the occurrence of clustered CPs. Mutant arr3 proteins with impaired binding to CL or AP-2 displayed reduced recruitment to CPs, but were still capable of inducing CP clustering. In contrast, though constitutively present in CPs, the COOH-terminal moiety of arr3, which contains CP binding sites but lacks receptor binding, did not induce CP clustering. Together, these results indicate that recruitment of functional arr3–GPCR complexes to CP is necessary to induce clustering. Latrunculin B or 16°C blocked CP rearrangements without affecting arr3 recruitment to CP. These results and earlier studies suggest that discrete CP zones exist on cell surfaces, each capable of supporting adjacent CPs, and that the cortical actin membrane skeleton is intimately involved with both the maintenance of existing CPs and the generation of new structures.

scholarly journals Role of G Protein-Coupled Receptor Kinases on the Agonist-Induced Phosphorylation and Internalization of the Follitropin Receptor

1999 ◽  
Vol 13 (6) ◽  
pp. 866-878 ◽  
Author(s):  
Maria de Fatima M. Lazari ◽  
Xuebo Liu ◽  
Kazuto Nakamura ◽  
Jeffrey L. Benovic ◽  
Mario Ascoli
Keyword(s):  
Cell Line ◽  
G Protein ◽  
Dominant Negative ◽  
Receptor Internalization ◽  
Dominant Negative Mutant ◽  
Deficient Mutant ◽  
G Protein Coupled Receptor ◽  
Western Blots ◽  
293 Cells ◽  
G Protein Coupled

Abstract The experiments presented herein were designed to identify members of the G protein-coupled receptor kinase (GRK) family that participate in the agonist-induced phosphorylation and internalization of the rat FSH receptor (rFSHR). Western blots of human kidney 293 cells (the cell line used in transfection experiments) and MSC-1 cells (a cell line derived from Sertoli cells that displays many of the differentiated functions of their normal counterparts) reveal the presence of GRK2 and GRK6 in both cell lines as well as GRK4 in MSC-1 cells. Cotransfection of 293 cells with the rFSHR and GRK2, GRK4α, or GRK6 resulted in an increase in the agonist-induced phosphorylation of the rFSHR. Cotransfections of the rFSHR with GRKs or arrestin-3 enhanced the agonist-induced internalization of the rFHSR, and combinations of GRKs and arrestin-3 were more effective than the individual components. To characterize the involvement of endogenous GRKs on phosphorylation and internalization, we inhibited endogenous GRK2 by overexpression of a kinase-deficient mutant of GRK2 or Gαt, a scavenger of Gβγ. We also inhibited endogenous GRK6 by overexpression of a kinase-deficient mutant of GKR6. All three constructs were effective inhibitors of phosphorylation, but only the kinase-deficient mutant of GRK2 and Gαt inhibited internalization. The inhibition of internalization induced by these two constructs was less pronounced than that induced by a dominant-negative mutant of the nonvisual arrrestins, however. The finding that inhibitors of GRK2 and GRK6 impair phosphorylation, but only the inhibitors of GRK2 impair internalization, suggests that different GRKs have differential effects on receptor internalization.

scholarly journals Dynamin and β-Arrestin Reveal Distinct Mechanisms for G Protein-coupled Receptor Internalization

1996 ◽  
Vol 271 (31) ◽  
pp. 18302-18305 ◽  
Author(s):  
Jie Zhang ◽  
Stephen S. G. Ferguson ◽  
Larry S. Barak ◽  
Luc Ménard ◽  
Marc G. Caron
Keyword(s):  
G Protein ◽  
Receptor Internalization ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

scholarly journals Structural assemblies of the di- and oligomeric G-protein coupled receptor TGR5 in live cells: an MFIS-FRET and integrative modelling study

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Annemarie Greife ◽  
Suren Felekyan ◽  
Qijun Ma ◽  
Christoph G. W. Gertzen ◽  
Lina Spomer ◽  
...  
Keyword(s):  
G Protein ◽  
Live Cells ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled ◽  
Modelling Study

Role of beta-Arrestin in Mediating Agonist-Promoted G Protein-Coupled Receptor Internalization

Science ◽  
1996 ◽  
Vol 271 (5247) ◽  
pp. 363-366 ◽  
Author(s):  
S. S. G. Ferguson ◽  
W. E. Downey ◽  
A.-M. Colapietro ◽  
L. S. Barak ◽  
L. M nard ◽  
...  
Keyword(s):  
G Protein ◽  
Receptor Internalization ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled
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