Constitutive GABAA Receptor Endocytosis Is Dynamin-mediated and Dependent on a Dileucine AP2 Adaptin-binding Motif within the β2 Subunit of the Receptor

Abstract

Receptor endocytosis is an important mechanism for regulating the synaptic efficacy of neurotransmitters. There is strong evidence that GABAA receptor endocytosis is clathrin-dependent; however, this process is not well understood. Here we demonstrate that in HEK 293 cells, endocytosis of GABAA receptors composed of either α1β2γ2Lor α1β2 subunits is blocked by the dominant negative dynamin construct K44A. Furthermore, we identify a dileucine AP2 adaptin-binding motif within the receptor β2 subunit that is critical for endocytosis. Internalization of GABAA receptors lacking this motif is dramatically inhibited, and the receptors appear the of receptors lacking the dileucine motif that is an in the of in HEK 293 receptors by of a the dileucine motif of the receptor β2 subunit not by a for the dileucine the dileucine motif of GABAA is the that an AP2 dileucine motif is critical for the endocytosis of this Receptor endocytosis is an important mechanism for regulating the synaptic efficacy of neurotransmitters. There is strong evidence that GABAA receptor endocytosis is clathrin-dependent; however, this process is not well understood. Here we demonstrate that in HEK 293 cells, endocytosis of GABAA receptors composed of either α1β2γ2Lor α1β2 subunits is blocked by the dominant negative dynamin construct K44A. Furthermore, we identify a dileucine AP2 adaptin-binding motif within the receptor β2 subunit that is critical for endocytosis. Internalization of GABAA receptors lacking this motif is dramatically inhibited, and the receptors appear the of receptors lacking the dileucine motif that is an in the of in HEK 293 receptors by of a the dileucine motif of the receptor β2 subunit not by a for the dileucine the dileucine motif of GABAA is the that an AP2 dileucine motif is critical for the endocytosis of this GABAA receptor is a by in in and of receptors in and is GABAA receptors the of and of GABAA receptor is a receptor composed of a and of subunits and receptors composed of subunits the GABAA receptor in the endocytosis is the of and is an important mechanism for the synaptic efficacy of GABAA receptors the mechanism is not well understood. of evidence that GABAA receptor endocytosis the of GABAA receptors in the of the receptor receptors and the and of GABAA receptors the AP2 that in of GABAA receptor endocytosis in HEK 293 that receptor endocytosis is blocked by a is not endocytosis a that GABAA receptor endocytosis in HEK 293 is blocked by is not blocked by the dynamin dominant negative that GABAA receptors a the by GABAA receptors not well Furthermore, the the receptor that in the process not the we the mechanism by GABAA receptors and identify the receptor and in an for and of of a and in the of for and of HEK 293 GABAA receptor by and and dynamin and and of Receptor the dileucine AP2 adaptin-binding within the GABAA receptor β2 the that the and of the and and and and by the subunits in dileucine and for the by 293 and HEK 293 cells, in by in of and in a of in a of the and in a for the and in for the of of we a that in HEK 293 GABAA receptors in of for by a of a for receptor endocytosis. identify the receptors that not this for in of an and by and receptors that the the not receptors that the of GABAA receptors in HEK 293 GABAA receptor endocytosis the HEK 293 GABAA receptors for by a a and and by of HEK 293 and an for and for and and and for of and and of of and and of and in and in for of the in HEK 293 HEK 293 in a the of an and the in synaptic an in the and a within of the endocytosis of GABAA HEK 293 receptors composed of subunits endocytosis is in the receptor not endocytosis and the by the of and the receptor is is by the within not of the receptors the receptor is the endocytosis the of endocytosis receptor the this however, of the is that the receptor endocytosis and that receptors the however, not receptors not the a of receptor endocytosis receptor receptor endocytosis is receptors composed of subunits dynamin of the dominant negative dynamin not of receptor endocytosis. for this in receptors a subunit not endocytosis of GABAA receptors in HEK 293 is blocked by the dominant negative dynamin K44A. for in and of GABAA receptors composed of either subunits by of dynamin not by dynamin for that endocytosis of the receptor the of the subunit endocytosis of receptors composed of subunits receptors composed of receptors lacking the subunit that the subunit is not for endocytosis. Furthermore, the endocytosis of receptors lacking the subunit by of dynamin not dynamin and in a subunit receptors not the by AP2 that dileucine and within of the receptor β2 subunit dileucine and a a in receptor the dileucine and and the and and the a of the in HEK 293 receptors composed of subunits and endocytosis for receptors and receptors composed of the motif is not for this the endocytosis of receptors lacking the dileucine composed of inhibited, that the dileucine motif is critical for this that of the dileucine motif in a of receptor we the of the receptors the in in Furthermore, the receptors by that the subunit the receptor in the of and of the for and the of the dileucine and AP2 adaptin-binding the GABAA receptor β2 motif dileucine motif of the dileucine AP2 adaptin-binding motif the GABAA receptor β2 subunit receptor endocytosis and HEK 293 by the receptor endocytosis and by Receptor endocytosis is in and AP2 motif Receptor endocytosis in receptors that the AP2 motif the β2 the of GABAA receptors composed of subunits by the receptor and and by of the dileucine AP2 adaptin-binding motif the GABAA receptor β2 subunit receptor endocytosis. for in Receptor endocytosis in receptors that the AP2 motif the β2 subunit receptors lacking the motif for lacking the dileucine AP2 adaptin-binding motif the β2 subunit in HEK 293 GABAA receptors composed of in the of and receptors and by the endocytosis of receptors lacking the dileucine motif the β2 subunit inhibited, we that the receptor the in an in the of of the the the of by in receptors the β2 subunit dileucine motif that for in for that of the receptor dileucine motif receptor in a that we a that the dileucine motif of the receptor β2 the dileucine of the subunit is a for AP2 the receptor for AP2 and receptor endocytosis. the either a the dileucine that for the dileucine in the of the a the the dileucine an in in in HEK 293 by a that the dileucine motif of the receptor β2 HEK 293 GABAA receptors composed of 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the receptor β2 subunit endocytosis. and dileucine GABAA receptor endocytosis. for in the of GABAA receptor endocytosis is not by dynamin dynamin not is evidence that GABAA receptors a is evidence the we demonstrate that GABAA receptor endocytosis is blocked by the dominant negative dynamin that is Furthermore, we that the AP2 dileucine motif the receptor β2 subunit is important for receptor a receptor subunit is not we that in HEK 293 and the of a that the dileucine motif the β2 subunit in the of GABAA receptor endocytosis in a that not the AP2 dileucine motif the receptor β2 is of and the in the dominant negative dynamin construct a for dynamin in GABAA receptor endocytosis. however, not the of in the of endocytosis is the of the AP2 the AP2 of GABAA receptor subunits dileucine and AP2 Here we demonstrate that of the dileucine motif the of the β2 subunit is critical for receptor endocytosis. and subunits of the and is that the GABAA receptor β2 subunit the subunit of AP2 of the of 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in HEK 293 cells, GABAA receptor endocytosis is and we an AP2 motif the receptor β2 subunit that is critical for this endocytosis. Furthermore, we that a the AP2 dileucine motif of the receptor β2 subunit in HEK 293 receptors and in that GABAA receptor is GABAA receptor and of the of the that GABAA receptor endocytosis. GABAA receptor is a by in in and of receptors in and is GABAA receptors the of and of GABAA receptor is a receptor composed of a and of subunits and receptors composed of subunits the GABAA receptor in the Receptor endocytosis is the of and is an important mechanism for the synaptic efficacy of GABAA receptors the mechanism is not well understood. of evidence that GABAA receptor endocytosis the of GABAA receptors in the of the receptor receptors and the and of GABAA receptors the AP2 that in of GABAA receptor endocytosis in HEK 293 that receptor endocytosis is blocked by a is not endocytosis a that GABAA receptor endocytosis in HEK 293 is blocked by is not blocked by the dynamin dominant negative that GABAA receptors a the by GABAA receptors not well Furthermore, the the receptor that in the process not the we the mechanism by GABAA receptors and identify the receptor and in an for and of of a and in the of for and of HEK 293 GABAA receptor by and and dynamin and and of Receptor the dileucine AP2 adaptin-binding within the GABAA receptor β2 the that the and of the and and and and by the subunits in dileucine and for the by 293 and HEK 293 cells, in by in of and in a of in a of the and in a for the and in for the of of we a that in HEK 293 GABAA receptors in of for by a of a for receptor endocytosis. identify the receptors that not this for in of an and by and receptors that the the not receptors that the of GABAA receptors in HEK 293 GABAA receptor endocytosis the HEK 293 GABAA receptors for by a a and and by of HEK 293 and an for and for and and and for of and and of of and and of and in and in for of the in HEK 293 HEK 293 in a the of an and the in synaptic an in the and a within of the and in an for and of of a and in the of for and of HEK 293 GABAA receptor by and and dynamin and and in an for and of of a and in the of for and of HEK 293 GABAA receptor by and and dynamin and and of Receptor the dileucine AP2 adaptin-binding within the GABAA receptor β2 the that the and of the and and and and by the subunits in dileucine and for the by the dileucine AP2 adaptin-binding within the GABAA receptor β2 the that the and of the and and and and by the subunits in dileucine and for the by HEK 293 and HEK 293 cells, in by in of and in a of in a of the and in a for the and in for the of of we a that in HEK 293 cells, in by in of and in a of in a of the and in a for the and in for the of of we a that in HEK 293 GABAA receptors in of for by a of a for receptor endocytosis. identify the receptors that not this for in of an and by and receptors that the the not receptors that the of GABAA receptors in HEK 293 GABAA receptor endocytosis the HEK 293 GABAA receptors for by a a and and by of HEK 293 and Receptor HEK 293 GABAA receptors in of for by a of a for receptor endocytosis. identify the receptors that not this for in of an and by and receptors that the the not receptors that the of GABAA receptors in HEK 293 GABAA receptor endocytosis Receptor the HEK 293 GABAA receptors for by a a and and by of HEK 293 and an for and for and and and for of and and an for and for and and and for of and and of of and and of and in and in for of of and and of and in and in for of the in HEK 293 HEK 293 in a the of an and the in synaptic an in the and a within of the of the in HEK 293 HEK 293 in a the of an and the in synaptic an in the and a within of the endocytosis of GABAA HEK 293 receptors composed of subunits endocytosis is in the receptor not endocytosis and the by the of and the receptor is is by the within not of the receptors the receptor is the endocytosis the of endocytosis receptor the this however, of the is that the receptor endocytosis and that receptors the however, not receptors not the a of receptor endocytosis receptor receptor endocytosis is receptors composed of subunits dynamin of the dominant negative dynamin not of receptor endocytosis. for this in receptors a subunit not that endocytosis of the receptor the of the subunit endocytosis of receptors composed of subunits receptors composed of receptors lacking the subunit that the subunit is not for endocytosis. 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Furthermore, the endocytosis of receptors lacking the subunit by of dynamin not dynamin and in a subunit receptors not the by AP2 that dileucine and within of the receptor β2 subunit dileucine and a a in receptor the dileucine and and the and and the a of the in HEK 293 receptors composed of subunits and endocytosis for receptors and receptors composed of the motif is not for this the endocytosis of receptors lacking the dileucine composed of inhibited, that the dileucine motif is critical for this that of the dileucine motif in a of receptor we the of the receptors the in in Furthermore, the receptors by that the subunit the receptor in the of and of the for and the endocytosis of receptors lacking the dileucine motif the β2 subunit inhibited, we that the receptor the in an in the of of the the the of by in receptors the β2 subunit dileucine motif that for in for that of the receptor dileucine motif receptor in a that we a that the dileucine motif of the receptor β2 the dileucine of the subunit is a for AP2 the receptor for AP2 and receptor endocytosis. the either a the dileucine that for the dileucine in the of the a the the dileucine an in in the in the HEK 293 the in the in HEK 293 cells, in the of the dileucine a in that an in in the dileucine the endocytosis of GABAA receptors that is that for the and of and however, the dileucine AP2 adaptin-binding motif is in and receptors and that of and is that in the of this we endocytosis in the of the of of dynamin GABAA receptor endocytosis and Furthermore, GABAA receptors that the dileucine AP2 adaptin-binding motif the β2 subunit in the of the GABAA receptors the of not is evidence that GABAA receptors a is evidence the we demonstrate that GABAA receptor endocytosis is blocked by the dominant negative dynamin that is Furthermore, we that the AP2 dileucine motif the receptor β2 subunit is important for receptor a receptor subunit is not we that in HEK 293 and the of a that the dileucine motif the β2 subunit in the of GABAA receptor endocytosis in a that not the AP2 dileucine motif the receptor β2 is of and the in the dominant negative dynamin construct a for dynamin in GABAA receptor endocytosis. however, not the of in the of endocytosis is the of the AP2 the AP2 of GABAA receptor subunits dileucine and AP2 Here we demonstrate that of the dileucine motif the of the β2 subunit is critical for receptor endocytosis. and subunits of the and is that the GABAA receptor β2 subunit the subunit of AP2 of the of and GABAA receptor subunits the and subunits of AP2 in the dileucine motif important for the endocytosis of a of this is the that an receptor this this of the AP2 motif the receptor β2 subunit not receptor endocytosis. the of this motif in the endocytosis of is that the motif not a in GABAA receptor endocytosis that is that not is that this motif AP2 subunits and of the receptor a mechanism for β2 of the motif β2 the the that GABAA receptor not a subunit within the is in the that receptors composed of either α1β2 subunits the that a subunit endocytosis that receptor endocytosis in the of a is that the subunit a in the of endocytosis receptor the subunit a dileucine motif in the of the motif is not for receptor endocytosis receptors that not the subunit endocytosis and the of this motif in receptors composed of subunits is not the not the that the dileucine motif the subunit is important this the dileucine motif the subunit is of a is that the of this the of the subunit AP2 of the and in the dileucine motif a in endocytosis a is in and GABAA receptors a the dileucine motif the receptor β2 that this the of AP2 the in a in receptor endocytosis and a in an in in HEK 293 and that the of receptors the a of receptor this the of the receptor by the of and by that the dileucine in that the in that dynamin GABAA receptor endocytosis of a that dynamin not GABAA receptor endocytosis. an a receptor endocytosis in HEK 293 the however, the of in the that of the endocytosis of the receptor a we endocytosis in the of dynamin GABAA receptor endocytosis. receptors that the dileucine motif the of for the the is that in the the receptor and endocytosis. of this we the of GABAA receptors in HEK 293 not appear GABAA receptor endocytosis in the that the by GABAA receptors the of the in the of that the of of receptor and the of receptor is an important receptor endocytosis. in this of we that the of dynamin not in receptor and the dileucine motif a and that the and that an by the we that in HEK 293 cells, GABAA receptor endocytosis is and we an AP2 motif the receptor β2 subunit that is critical for this endocytosis. Furthermore, we that a the AP2 dileucine motif of the receptor β2 subunit in HEK 293 receptors and in that GABAA receptor is GABAA receptor and of the of the that GABAA receptor endocytosis. is evidence that GABAA receptors a is evidence the we demonstrate that GABAA receptor endocytosis is blocked by the dominant negative dynamin that is Furthermore, we that the AP2 dileucine motif the receptor β2 subunit is important for receptor a receptor subunit is not we that in HEK 293 and the of a that the dileucine motif the β2 subunit in the of GABAA receptor endocytosis in a that not the AP2 dileucine motif the receptor β2 is of and the in the dominant negative dynamin construct a for dynamin in GABAA receptor endocytosis. however, not the of in the of endocytosis is the of the AP2 the AP2 of GABAA receptor subunits dileucine and AP2 Here we demonstrate that of the dileucine motif the of the β2 subunit is critical for receptor endocytosis. and subunits of the and is that the GABAA receptor β2 subunit the subunit of AP2 of the of and GABAA receptor subunits the and subunits of AP2 in the dileucine motif important for the endocytosis of a of this is the that an receptor this this of the AP2 motif the receptor β2 subunit not receptor endocytosis. the of this motif in the endocytosis of is that the motif not a in GABAA receptor endocytosis that is that not is that this motif AP2 subunits and of the receptor a mechanism for β2 of the motif β2 the the that GABAA receptor not a subunit within the is in the that receptors composed of either α1β2 subunits the that a subunit endocytosis that receptor endocytosis in the of a is that the subunit a in the of endocytosis receptor endocytosis. the subunit a dileucine motif in the of the motif is not for receptor endocytosis receptors that not the subunit endocytosis and the of this motif in receptors composed of subunits is not the not the that the dileucine motif the subunit is important this the dileucine motif the subunit is of a is that the of this the of the subunit AP2 of the and in the dileucine motif a in endocytosis a is in and GABAA receptors a the dileucine motif the receptor β2 that this the of AP2 the in a in receptor endocytosis and a in an in in HEK 293 and that the of receptors the a of receptor this the of the receptor by the of and by that the dileucine in that the in that dynamin GABAA receptor endocytosis of a that dynamin not GABAA receptor endocytosis. an a receptor endocytosis in HEK 293 the however, the of in the that of the endocytosis of the receptor a we endocytosis in the of dynamin GABAA receptor endocytosis. receptors that the dileucine motif the of for the the is that in the the receptor and endocytosis. of this we the of GABAA receptors in HEK 293 not appear GABAA receptor endocytosis in the that the by GABAA receptors the of the in the of that the of of receptor and the of receptor is an important receptor endocytosis. in this of we that the of dynamin not in receptor and the dileucine motif a and that the and that an by the Here we that in HEK 293 cells, GABAA receptor endocytosis is and we an AP2 motif the receptor β2 subunit that is critical for this endocytosis. Furthermore, we that a the AP2 dileucine motif of the receptor β2 subunit in HEK 293 receptors and in that GABAA receptor is GABAA receptor and of the of the that GABAA receptor endocytosis.