Clathrin-independent endocytosis of GABA(A) receptors in HEK 293 cells.
The endocytosis of GABA(A) receptors was investigated in HEK 293 cells expressing receptor alpha1beta2- and alpha1beta2gamma2-subunit combinations. For assessment of internalized receptors by radioimmunoassay or immunofluorescence, a triple c-myc epitope was introduced into the amino terminus of the beta2 subunit. An assay based on biotin inaccessibility was used for alpha1 subunits. GABA(A) alpha1beta2- and alpha1beta2gamma2-subunit receptors were internalized with a t(1/2) of 5.5 min at 37 degrees C. With both subunit combinations, phorbol 12-myristate 3-acetate enhanced internalization by nearly 100%. Treatment of the cells with hypertonic sucrose prevented both the basal and phorbol ester-induced endocytosis of GABA(A) receptors. GF 109203X, an inhibitor of protein kinase C, blocked the stimulation by phorbol ester but had no detectable effect on basal receptor endocytosis. Coexpression with a dominant-negative mutant of dynamin (K44A) led to a 100% enhancement of GABA(A) receptor internalization, while the endocytosis of beta(2)-adrenergic receptors was completely prevented. The results indicate that the endocytosis of GABA(A) alpha1beta2-subunit receptors in HEK cells is constitutive, positively modulated by activation of protein kinase C, and occurs by a mechanism that requires neither the participation of a GABA(A) receptor gamma2 subunit nor a clathrin-mediated pathway.
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Division of Neuroscience and Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Barnes
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D000818:Animals; D002460:Cell Line / Q000201:enzymology / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D002645:Chickens; D002966:Clathrin / Q000502:physiology*; D034281:Dynamins; D004705:Endocytosis / Q000187:drug effects / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology; D011963:Receptors, GABA-A / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D002966:Clathrin; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D011493:Protein Kinase C; D020558:GTP Phosphohydrolases; D034281:Dynamins; D013755:Tetradecanoylphorbol Acetate