scholarly journals The Predicted G-Protein-Coupled Receptor GPR-1 Is Required for Female Sexual Development in the Multicellular Fungus Neurospora crassa

2006 ◽  
Vol 5 (9) ◽  
pp. 1503-1516 ◽  
Author(s):  
Svetlana Krystofova ◽  
Katherine A. Borkovich
Keyword(s):  
Neurospora Crassa ◽  
G Protein ◽  
Sexual Development ◽  
Fluorescent Protein ◽  
Protein Fusion ◽  
Phenotypic Analysis ◽  
Reproductive Structures ◽  
Epistasis Analysis ◽  
Green Fluorescent ◽  
G Protein Coupled

ABSTRACTG-protein-coupled receptors (GPCRs) control important aspects of asexual and sexual development in eukaryotic organisms. We have identified a predicted GPCR in the filamentous fungusNeurospora crassawith similarity to cyclic AMP-receptor like GPCRs fromDictyostelium discoideumand GCR1 fromArabidopsis thaliana. Expression ofgpr-1is highest in female reproductive structures, and deletion ofgpr-1leads to defects during sexual development. Unfertilized female structures (protoperithecia) fromΔgpr-1strains are weakly pigmented, small, and submerged in the agar. The perithecia produced after fertilization have deformed beaks that lack ostioles, the openings through which ascospores are discharged. Localization studies using a GPR-1-green fluorescent protein fusion protein showed that GPR-1 is targeted to female reproductive structures. Genetic epistasis experiments with the three Gα genes were inconclusive due to the early block in mating exhibited by Δgna-1strains. Phenotypic analysis of mutants from a high-throughputN. crassaknockout project allowed identification of BEK-1, a homeodomain transcription factor that is a potential target of GPR-1. The perithecial defects ofΔbek-1strains are similar to those of theΔgpr-1strain, and epistasis analysis indicates thatbek-1could function downstream ofgpr-1during postfertilization events. The effect must be posttranscriptional, asbek-1transcript levels are not affected inΔgpr-1strains. The lack of ostioles inΔgpr-1and Δbek-1mutants has an undesirable effect on the ability to spread progeny (ascospores) by the normal ejection mechanism and would severely compromise the fitness of these strains in nature.

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 Genetic analysis of the role of G protein–coupled receptor signaling in electrotaxis

2002 ◽  
Vol 157 (6) ◽  
pp. 921-928 ◽  
Author(s):  
Min Zhao ◽  
Tian Jin ◽  
Colin D. McCaig ◽  
John V. Forrester ◽  
Peter N. Devreotes
Keyword(s):  
G Protein ◽  
Fluorescent Protein ◽  
Electrical Potential ◽  
Leading Edge ◽  
Receptor Signaling ◽  
Protein Signaling ◽  
G Protein Coupled Receptor ◽  
Camp Receptor ◽  
Green Fluorescent ◽  
G Protein Coupled

Cells display chemotaxis and electrotaxis by migrating directionally in gradients of specific chemicals or electrical potential. Chemotaxis in Dictyostelium discoideum is mediated by G protein–coupled receptors. The unique Gβ is essential for all chemotactic responses, although different chemoattractants use different receptors and Gα subunits. Dictyostelium amoebae show striking electrotaxis in an applied direct current electric field. Perhaps electrotaxis and chemotaxis share similar signaling mechanisms? Null mutation of Gβ and cAMP receptor 1 and Gα2 did not abolish electrotaxis, although Gβ-null mutations showed suppressed electrotaxis. By contrast, G protein signaling plays an essential role in chemotaxis. G protein–coupled receptor signaling was monitored with PHcrac–green fluorescent protein, which translocates to inositol phospholipids at the leading edge of cells during chemotaxis. There was no intracellular gradient of this protein during electrotaxis. However, F-actin was polymerized at the leading edge of cells during electrotaxis. We conclude that reception and transduction of the electrotaxis signal are largely independent of G protein–coupled receptor signaling and that the pathways driving chemotaxis and electrotaxis intersect downstream of heterotrimeric G proteins to invoke cytoskeletal elements.

scholarly journals Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Yoon Namkung ◽  
Christian Le Gouill ◽  
Viktoria Lukashova ◽  
Hiroyuki Kobayashi ◽  
Mireille Hogue ◽  
...  
Keyword(s):  
G Protein ◽  
Fluorescent Protein ◽  
Protein Translocation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Specific Cell ◽  
Cell Compartments ◽  
Green Fluorescent ◽  
G Protein Coupled

Abstract Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however, robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and β-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligand-promoted recruitment or sequestration of RLuc-tagged proteins to, or from, specific cell compartments, as well as sensitive subcellular BRET imaging for protein translocation visualization. These sensors are scalable to high-throughput formats and allow quantitative pharmacological studies of GPCR trafficking in real time, in live cells, revealing ligand-dependent biased trafficking of receptor/β-arrestin complexes.

Sign in / Sign up

Export Citation Format

Share Document