Visualization of a functionally enhanced GFP-tagged galanin R2 receptor in PC12 cells: Constitutive and ligand-induced internalization

Abstract

Trafficking of the galanin R2 receptor (GALR2) fused with enhanced GFP (EGFP) was studied by using confocal fluorescence microscopy. The fusion protein was predominantly localized on the plasma membrane with some intracellular fluorescent structures (vesicles), mainly in the perinuclear region. Incubation with galanin resulted in a concentration-dependent increase in intracellular Ca 2؉ concentration levels, suggesting that the GALR2-EGFP conjugate is functional. After blocking endocytosis with methyl-␤-cyclodextrin GALR2-EGFP expression was increased on the surface and decreased in the cytoplasm. Blocking endocytic recycling with monensin caused an increase of intracellular GALR2-EGFP accumulation and a decrease of fluorescence on the plasma membrane. GALR2-EGFP on the plasma membrane was internalized within 5-10 min after treatment with galanin or AR-M1896, a selective GALR2 agonist, with a dramatic reduction in plasma membrane localization and appearance in intracellular vesicles. Neither M35 nor M40, two galanin analogues with putative antagonistic action, prevented GALR2 agonist-induced internalization of GALR2-EGFP, suggesting that they are not antagonists at this receptor under the present circumstances. Galanin stimulation at low temperature caused GALR2-EGFP aggregation and clustering on the surface but no translocation to cytoplasm. After coincubation with galanin the GALR2-EGFP was colocalized with internalized Texas red-transferrin, a marker of the clathrin endocytic pathway. Hyperosmotic sucrose inhibited internalization of GALR2-EGFP. Taken together these findings indicate that GALR2 undergoes constitutive endocytosis and recycling and that both ligand-independent and liganddependent internalization use the clathrin-dependent endocytic recycling pathway.

Visualizing odorant receptor trafficking in living cells down to the single-molecule level

“…Although constitutive internalization in the absence of a ligand has been reported for several GPCRs (19,(24)(25)(26)(27)(28), to our knowledge no indication of this phenomenon has been provided for ORs. Our present results indicate that receptor internalization, which is an agonist-dependent process in the classical model of GPCR signaling, can also occur without G protein activation and suggest that GPCRs can adopt many more conformational states than is usually believed (29).…”

Global Ablation of the Mouse Rab11a Gene Impairs Early Embryogenesis and Matrix Metalloproteinase Secretion

“…This effect resembled wild-type MEFs treated by monensin, an inhibitor of the recycling trafficking (53,54). Furthermore, the partially restored MMP2 secretion in rescued Rab11a null MEFs was sensitive to the blockage by monensin (Fig.…”

Physiology, Signaling, and Pharmacology of Galanin Peptides and Receptors: Three Decades of Emerging Diversity

“…Studies using cell lines transfected with galanin receptors have demonstrated GAL 1 homodimerization and internalization (Wang et al, 1998b;Xia et al, 2004Xia et al, , 2005bXia et al, , 2008Wirz et al, 2005). GAL 2 also undergoes internalization upon ligand binding (Xia et al, 2004(Xia et al, , 2005b.…”

“…This allowed the mapping of galanin receptor transcripts using Northern blotting, qRT-PCR (Waters and Krause, 2000), and in situ hybridization (O'Donnell et al, 1999Burazin et al, 2000;Mennicken et al, 2002;Le Maître et al, 2013). Thus far, no totally specific and reliable antigalanin receptor antibodies have been generated (Lu and Bartfai, 2009), so the exact regional and cellular localization of the three galanin receptor proteins in brain and other tissues remains to be elucidated, although studies of tagged receptors in transfected cell lines have provided some information on trafficking of GAL 1 and GAL 2 (Xia et al, 2004Wirz et al, 2005).…”