Mechanisms of homomeric alpha1 glycine receptor endocytosis.
Little is known regarding the mechanism(s) by which glycine receptors are endocytosed. Here we examined the endocytosis of homomeric alpha1 glycine receptors expressed in HEK 293 cells using immunofluorescence/confocal microscopy and whole-cell patch-clamp recordings. Our studies demonstrate that constitutive endocytosis of glycine receptors is blocked by the dominant negative dynamin construct K44A and that intracellular dialysis with peptide P4, a dynamin/amphiphysin-disrupting peptide, increased whole-cell glycine-gated chloride currents. To examine whether receptor endocytosis could be regulated by PKC, experiments with the PKC activator PMA (phorbol 12-myristate 13-acetate) were performed. PMA, but not its inactive analogue PMM (phorbol 12-monomyristate), stimulated receptor endocytosis and inhibited glycine-gated chloride currents. Similar to constitutive endocytosis, PKC-stimulated endocytosis was blocked by dynamin K44A. Mutation of a putative AP2 adaptin dileucine motif (L314A, L315A) present in the receptor cytoplasmic loop blocked PMA-stimulated receptor endocytosis and also prevented PMA inhibition of glycine receptor currents. In patch-clamp experiments, intracellular dialysis of a 12-amino acid peptide corresponding to the region of the receptor containing the dileucine motif prevented PKC modulation of wild-type glycine receptors. Unlike PKC modulation of the receptor, constitutive endocytosis was not affected by mutation of this dileucine motif. These results demonstrate that PKC activation stimulates glycine receptor endocytosis, that both constitutive endocytosis and PKC-stimulated endocytosis are dynamin-dependent, and that PKC-stimulated endocytosis, but not constitutive endocytosis, occurs via the dileucine motif (L314A, L315A) within the cytoplasmic loop of the receptor.
Huang
Renqi
R
Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, Texas 76107, USA.
He
Shaoqing
S
Chen
Zhenglan
Z
Dillon
Glenn H
GH
Leidenheimer
Nancy J
NJ
D002460:Cell Line; D034281:Dynamins / Q000502:physiology; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal; D018408:Patch-Clamp Techniques; D011493:Protein Kinase C / Q000378:metabolism; D018009:Receptors, Glycine / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D018009:Receptors, Glycine; D011493:Protein Kinase C; D034281:Dynamins; D013755:Tetradecanoylphorbol Acetate