Uncoupling and endocytosis of 5-hydroxytryptamine 4 receptors. Distinct molecular events with different GRK2 requirements.
The 5-hydroxytryptamine type 4 receptors (5-HT4Rs) are involved in memory, cognition, feeding, respiratory control, and gastrointestinal motility through activation of a G(s)/cAMP pathway. We have shown that 5-HT4R undergoes rapid and profound homologous uncoupling in neurons. However, no significant uncoupling was observed in COS-7 or HEK293 cells, which expressed either no or a weak concentration of GRK2, respectively. High expression of GRK2 in neurons is likely to be the reason for this difference because overexpression of GRK2 in COS-7 and HEK293 cells reproduced rapid and profound uncoupling of 5-HT4R. We have also shown, for the first time, that GRK2 requirements for uncoupling and endocytosis were very different. Indeed, beta-arrestin/dynamin-dependent endocytosis was observed in HEK293 cells without any need of GRK2 overexpression. In addition to this difference, uncoupling and beta-arrestin/dynamin-dependent endocytosis were mediated through distinct mechanisms. Neither uncoupling nor beta-arrestin/dynamin-dependent endocytosis required the serine and threonine residues localized within the specific C-terminal domains of the 5-HT4R splice variants. In contrast, a cluster of serines and threonines, common to all variants, was an absolute requirement for beta-arrestin/dynamin-dependent receptor endocytosis, but not for receptor uncoupling. Furthermore, beta-arrestin/dynamin-dependent endocytosis and uncoupling were dependent on and independent of GRK2 kinase activity, respectively. These results clearly demonstrate that the uncoupling and endocytosis of 5-HT4R require different GRK2 concentrations and involve distinct molecular events.
Barthet
Gaël
G
CNRS UMR5203, Montpellier, F-34094, France.
Gaven
Florence
F
Framery
Bérénice
B
Shinjo
Katsuhiro
K
Nakamura
Takaaki
T
Claeysen
Sylvie
S
Bockaert
Joël
J
Dumuis
Aline
A
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000502:physiology*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D034281:Dynamins / Q000378:metabolism; D004705:Endocytosis; D004797:Enzyme-Linked Immunosorbent Assay; D005799:Genes, Dominant; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D044422:Receptors, Serotonin, 5-HT4 / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases; D000071557:beta-Arrestins
D019390:Arrestins; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D012333:RNA, Messenger; D000071557:beta-Arrestins; D044422:Receptors, Serotonin, 5-HT4; D013912:Threonine; D012694:Serine; D012313:RNA; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D034281:Dynamins