Cell Surface Expression of 5-Hydroxytryptamine Type 3 Receptors Is Promoted by RIC-3

Abstract

RIC-3 has been identified as a molecule essential for the recruitment of functional nicotinic acetylcholine receptors composed of α7, but it exhibits inhibitory effects on α4β2 or α3β4 receptors. In this study, we investigated the role of RIC-3 in the recruitment of 5-hydroxytryptamine type 3A (5-HT3A) receptors to the cell surface. Although RIC-3 is not essential for the surface transport of 5-HT3A receptors, we found that its presence enhances both receptor transport and function in a concentration-dependent manner. RIC-3 is localized to the endoplasmic reticulum, as evidenced by co-localization with the chaperone molecule, binding protein (BiP). RIC-3 is not detected at significant levels on the cell surface when expressed alone or in the presence of 5-HT3A. RIC-3 and 5-HT3A show a low level interaction that is transient (<4 h). That RIC-3 can interact with an endoplasmic reticulum-retained 5-HT3A construct, combined with the transient interaction observed and lack of significant surface-expressed that RIC-3 a role in 5-HT3A receptor or transport to the cell surface. RIC-3 has been identified as a molecule essential for the recruitment of functional nicotinic acetylcholine receptors composed of α7, but it exhibits inhibitory effects on α4β2 or α3β4 receptors. In this study, we investigated the role of RIC-3 in the recruitment of 5-hydroxytryptamine type 3A (5-HT3A) receptors to the cell surface. Although RIC-3 is not essential for the surface transport of 5-HT3A receptors, we found that its presence enhances both receptor transport and function in a concentration-dependent manner. RIC-3 is localized to the endoplasmic reticulum, as evidenced by co-localization with the chaperone molecule, binding protein (BiP). RIC-3 is not detected at significant levels on the cell surface when expressed alone or in the presence of 5-HT3A. RIC-3 and 5-HT3A show a low level interaction that is transient (<4 h). That RIC-3 can interact with an endoplasmic reticulum-retained 5-HT3A construct, combined with the transient interaction observed and lack of significant surface-expressed that RIC-3 a role in 5-HT3A receptor or transport to the cell surface. of the is not the but the of in not and of but can of is and is to at the of the of the for and or or receptor in is receptor is of been identified as in and and to in of 5-hydroxytryptamine type endoplasmic acetylcholine nicotinic binding binding 5-hydroxytryptamine type endoplasmic acetylcholine nicotinic binding binding receptors to the of that the nicotinic and receptors. of the of this that and transport of receptors the of and the endoplasmic been identified in receptors for and acetylcholine of receptors the a for surface with the of the by the chaperone In the receptors been identified as that protein the to in by the to the chaperone found in the a of chaperone has been the for the recruitment of the of the of receptor and surface and in receptor and and RIC-3 and in receptor transport and protein and and in and surface and and and protein and protein the of this and an for on of receptor molecule has been in the recruitment of receptors. RIC-3 has been to receptor we the role of RIC-3 on 5-HT3A transport to the cell surface. RIC-3 identified in a for that to function of RIC-3 that it is for the of functional of RIC-3 to the and not at combined with the that to the cell in the of RIC-3 is of a role in transport the to the surface. a that RIC-3 is not in the transport of receptors to the cell surface. the function of RIC-3 to at the level of protein and is essential for the of the binding RIC-3 found at the cell surface with the the of RIC-3 chaperone investigated the role of RIC-3 on the of 5-HT3A receptors and a significant role in the of receptor recruitment to the cell surface. interaction of RIC-3 with 5-HT3A the and is and RIC-3 is not detected at the cell surface. that RIC-3 a role in the transport of 5-HT3A receptors, by protein and and in with and in an of by of for with of 5-HT3A expressed the and with the or the and in by as RIC-3 a and by and as or on protein to RIC-3 in an on RIC-3 is expressed on the cell or an as the for and for RIC-3 a significant by on and and at and at in in and in for and in and surface by the of and the the the and to surface or receptor a of of and of at receptor in in in surface in the of and levels for in and in for in an and as the with at the of to for to for in for at of of by the and to the and to the is the of at a of for with for in in the presence of for in and as and by by in and cell surface for on by in in and at and protein in the of 5-HT3A receptors and in of of and the 5-HT3A the of or to the of 5-HT3A and of the the to or functional effects of has been that can the cell surface and function as a In of this surface of the can detected in the of it is in a is In receptors to as observed the of receptors observed in an In of presence in transport the of and a cell surface of is observed a to is to that to is a of a In to surface a significant in is observed in the in cell surface levels of we to the of expressed RIC-3 the the on to the that as for and that the is a the in this is to in In the presence of a level of RIC-3 is a as as an surface In of this low levels of surface to levels in RIC-3 to the for an RIC-3 and show but not and has been that RIC-3 is localized to the in a cell the nicotinic acetylcholine receptors RIC-3 localized when 5-HT3A the of RIC-3 with significant surface or the presence of surface protein for RIC-3 not that RIC-3 the in the or transport of 5-HT3A receptors as a we a transient presence of RIC-3 at the cell of 5-HT3A and RIC-3 by RIC-3 and and the of 5-HT3A or RIC-3 as protein RIC-3 as and RIC-3 and for the in receptors on a and by RIC-3 and or RIC-3 and RIC-3 by of the RIC-3 surface of and of of with by the of the RIC-3 the cell surface observed by we the cell to cell surface and receptor is to that an is to is not to with an of a as the of receptors is in the presence of In for cell surface receptors cell been of RIC-3 with of In of a role in the recruitment of 5-HT3A receptors to the cell surface by cell surface levels of as RIC-3 significant at or with effects at of the surface and receptor levels this and that the transport for a in receptor significant when of RIC-3 that RIC-3 a on of levels in the presence of of with of and of RIC-3 receptor to that of in receptor levels detected to by to in the of receptor levels as in the presence of this the of at in at significant the RIC-3 the of RIC-3 on 5-HT3A receptor we a and a 5-HT3A with of RIC-3 to a of to a for of In of the observed with of RIC-3 the for for the to as for 5-HT3A with an in cell surface receptor and a lack of in the of of receptor by in the presence of of with of and of RIC-3 receptor in to of with the and to of and for an of the to the of at in the presence of and of for not with an of significant the RIC-3 the of protein and we on In with and with 5-HT3A observed the of an of observed with the identified when the RIC-3 to RIC-3 this not to in the RIC-3 in a to both that the interaction is observed when the is the RIC-3 receptor the and this is the for it that interaction at this that to interact the we the a 5-HT3A with an that has been to in the of this to for the that the interaction can the it is that the interaction is its transport and the the and RIC-3 interaction is we and RIC-3 with and and in the presence of for this of we found that the level the of RIC-3 that the interaction 5-HT3A and RIC-3 is the of RIC-3 at the cell surface when with the nicotinic acetylcholine in we the for 5-HT3A and cell surface of RIC-3 or RIC-3 and surface and for the of RIC-3 by show that RIC-3 not at the cell surface when expressed alone with 5-HT3A of cell surface with to that the been identified in a in for of a in the acetylcholine receptor to the of a that is for to In this in RIC-3 found to this an essential role for RIC-3 in acetylcholine receptor by in to and but not in RIC-3 localized to cell as receptors in an essential role for RIC-3 in the on the of this role of RIC-3 to receptors an inhibitory of RIC-3 observed on the function of α4β2 and α3β4 and receptors, with on receptors. 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RIC-3 at the cell surface by receptors. this is not the for 5-HT3A receptors, in with the low transient interaction that can significant levels of surface RIC-3 not when with 5-HT3A. low level of 5-HT3A and RIC-3 is with a transient or low to and the interaction to the of of RIC-3 on 5-HT3A a to 5-HT3A receptor we observed that the in 5-HT3A receptor surface to the level of a by functional an in receptor levels when RIC-3 in is to of a of the 5-HT3A been and receptor is the receptor the is by RIC-3 not this with a role for RIC-3 in 5-HT3A receptor by a as receptor observed for or receptors to and to the cell surface to the observed for we the of surface to receptors, it that the in cell surface levels at low levels for 3A to of the of in receptor the of RIC-3 on both surface and 5-HT3A receptor the of RIC-3 the cell and the low level interaction with 5-HT3A receptors that RIC-3 that is a for protein and transport to the cell surface. In to in RIC-3 is not an for the surface of 5-HT3A in as it is not expressed to the in the or of receptor function is that RIC-3 a role in receptor by the of receptor a that is a for or a to with receptor to in of the is not the but the of in not and of but can of is and is to at the of the of the for and or or receptor in is receptor is of been identified as in and and to in of 5-hydroxytryptamine type endoplasmic acetylcholine nicotinic binding binding 5-hydroxytryptamine type endoplasmic acetylcholine nicotinic binding binding receptors to the of that the nicotinic and receptors. of the of this that and transport of receptors the of and the endoplasmic been identified in receptors for and acetylcholine of receptors the a for surface with the of the by the chaperone In the receptors been identified as that protein the to in by the In to the chaperone found in the a of chaperone has been the for the recruitment of the of the of receptor and surface and in receptor and and RIC-3 and in receptor transport and protein and and in and surface and and and protein and protein the of this and an for on of receptor molecule has been in the recruitment of receptors. RIC-3 has been to receptor we the role of RIC-3 on 5-HT3A transport to the cell surface. RIC-3 identified in a for that to function of RIC-3 that it is for the of functional of RIC-3 to the and not at combined with the that to the cell in the of RIC-3 is of a role in transport the to the surface. a that RIC-3 is not in the transport of receptors to the cell surface. the function of RIC-3 to at the level of protein and is essential for the of the binding RIC-3 found at the cell surface with the the of RIC-3 chaperone investigated the role of RIC-3 on the of 5-HT3A receptors and a significant role in the of receptor recruitment to the cell surface. interaction of RIC-3 with 5-HT3A the and is and RIC-3 is not detected at the cell surface. that RIC-3 a role in the transport of 5-HT3A receptors, by protein and and in with and in an of by of for with of 5-HT3A expressed the and with the or the and in by as RIC-3 a and by and as or on protein to RIC-3 in an on RIC-3 is expressed on the cell or an as the for and for RIC-3 a significant by on and and at and at in in and in for and in and surface by the of and the the the and to surface or receptor a of of and of at receptor in in in surface in the of and levels for in and in for in an and as the with at the of to for to for in for at of of by the and to the and to the is the of at a of for with for in in the presence of for in and as and by by in and cell surface for on by in in and at and protein in and and in with and in an of by of for with of 5-HT3A expressed the and with the or the and in by as RIC-3 a and by and as or on protein to RIC-3 in an on RIC-3 is expressed on the cell or an as the for and for RIC-3 a significant by on and and at and at in in and in for and in and surface by the of and the the the and to surface or receptor a of of and of at receptor in in in surface in the of and levels for in and in for in an and as the with at the of to for to for in for at of of by the and to the and to the is the of at a of for with for in in the presence of for in and as and by by in and cell surface for on by in in and at and protein in the of 5-HT3A receptors and in of of and the 5-HT3A the of or to the of 5-HT3A and of the the to or functional effects of has been that can the cell surface and function as a In of this surface of the can detected in the of it is in a is In receptors to as observed the of receptors observed in an In of presence in transport the of and a cell surface of is observed a to is to that to is a of a In to surface a significant in is observed in the in cell surface levels of we to the of expressed RIC-3 the the on to the that as for and that the is a the in this is to in In the presence of a level of RIC-3 is a as as an surface In of this low levels of surface to levels in RIC-3 to the for an RIC-3 and show but not and has been that RIC-3 is localized to the in a cell the nicotinic acetylcholine receptors RIC-3 localized when 5-HT3A the of RIC-3 with significant surface or the presence of surface protein for RIC-3 not that RIC-3 the in the or transport of 5-HT3A receptors as a we a transient presence of RIC-3 at the cell the cell surface observed by we the cell to cell surface and receptor is to that an is to is not to with an of a as the of receptors is in the presence of In for cell surface receptors cell been of RIC-3 with of In of a role in the recruitment of 5-HT3A receptors to the cell surface by cell surface levels of as RIC-3 significant at or with effects at of the surface and receptor levels this and that the transport for a in receptor significant when of RIC-3 that RIC-3 a on of levels in the presence of of with of and of RIC-3 receptor to that of in receptor levels detected to by to in the of receptor levels as in the presence of this the of at in at significant the RIC-3 the of RIC-3 on 5-HT3A receptor we a and a 5-HT3A with of RIC-3 to a of to a for of In of the observed with of RIC-3 the for for the to as for 5-HT3A with an in cell surface receptor and a lack of in the of of receptor by in the presence of of with of and of 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surface with to that the been the of 5-HT3A receptors and in of of and the 5-HT3A the of or to the of 5-HT3A and of the the to or functional effects of has been that can the cell surface and function as a In of this surface of the can detected in the of it is in a is In receptors to as observed the of receptors observed in an In of presence in transport the of and a cell surface of is observed a to is to that to is a of a In to surface a significant in is observed in the in cell surface levels of we to the of expressed RIC-3 the the on to the that as for and that the is a the in this is to in In the presence of a level of RIC-3 is a as as an surface In of this low levels of surface to levels in RIC-3 to the for an RIC-3 and show but not and has been that RIC-3 is localized to the in a cell the nicotinic acetylcholine receptors RIC-3 localized when 5-HT3A the of RIC-3 with significant surface or the presence of surface protein for RIC-3 not that RIC-3 the in the or transport of 5-HT3A receptors as a we a transient presence of RIC-3 at the cell surface. the cell surface observed by we the cell to cell surface and receptor is to that an is to is not to with an of a as the of receptors is in the presence of In for cell surface receptors cell been of RIC-3 with of In of a role in the recruitment of 5-HT3A receptors to the cell surface by cell surface levels of as RIC-3 significant at or with effects at of the surface and receptor levels this and that the transport for a in receptor significant when of RIC-3 that RIC-3 a on 5-HT3A. the of RIC-3 on 5-HT3A receptor we a and a 5-HT3A with of RIC-3 to a of to a for of In of the observed with of RIC-3 the for for the to as for 5-HT3A with an in cell surface receptor and a lack of in the of the of protein and we on In with and with 5-HT3A observed the of an of observed with the identified when the RIC-3 to RIC-3 this not to in the RIC-3 in a to both that the interaction is observed when the is the RIC-3 receptor the and this is the for it that interaction at this that to interact the we the a 5-HT3A with an that has been to in the of this to for the that the interaction can the it is that the interaction is its transport and the the and RIC-3 interaction is we and RIC-3 with and and in the presence of for this of we found that the level the of RIC-3 that the interaction 5-HT3A and RIC-3 is the of RIC-3 at the cell surface when with the nicotinic acetylcholine in we the for 5-HT3A and cell surface of RIC-3 or RIC-3 and surface and for the of RIC-3 by show that RIC-3 not at the cell surface when expressed alone with 5-HT3A of cell surface with to that the been identified in a in for of a in the acetylcholine receptor to the of a that is for to In this in RIC-3 found to this an essential role for RIC-3 in acetylcholine receptor by in to and but not in RIC-3 localized to cell as receptors in an essential role for RIC-3 in the on the of this role of RIC-3 to receptors an inhibitory of RIC-3 observed on the function of α4β2 and α3β4 and receptors, with on receptors. In to the for RIC-3 on transport the cell identified role for RIC-3 in the transport of receptors to the cell surface. a role in the of the binding Although this role the and a for of receptors to the cell the of this that surface in the of RIC-3 and that the on the cell the for RIC-3 in the transport of and the of the inhibitory function of RIC-3 on α4β2 and α3β4 and receptors and the lack of of RIC-3 on or receptors we to the role of RIC-3 on and 5-HT3A receptor transport to the cell surface. in with for receptor function we found role for RIC-3 on the surface transport of receptors not In to the of RIC-3 on receptor we observed a of 5-HT3A receptor surface and is not but in the or of the 5-HT3A we observed in cell surface in the presence of to the of 5-HT3A to in is the of surface of a in the cell type has been when at levels is a in the transport of cell surface In with a role for RIC-3 this RIC-3 exhibits co-localization with the we not significant levels of RIC-3 at the cell surface by or cell surface to the observed an is that in the study, receptors. RIC-3 at the cell surface by receptors. this is not the for 5-HT3A receptors, in with the low transient interaction that can significant levels of surface RIC-3 not when with 5-HT3A. low level of 5-HT3A and RIC-3 is with a transient or low to and the interaction to the of of RIC-3 on 5-HT3A a to 5-HT3A receptor we observed that the in 5-HT3A receptor surface to the level of a by functional an in receptor levels when RIC-3 in is to of a of the 5-HT3A been and receptor is the receptor the is by RIC-3 not this with a role for RIC-3 in 5-HT3A receptor by a as receptor observed for or receptors to and to the cell surface to the observed for we the of surface to receptors, it that the in cell surface levels at low levels for 3A to of the of in receptor the of RIC-3 on both surface and 5-HT3A receptor the of RIC-3 the cell and the low level interaction with 5-HT3A receptors that RIC-3 that is a for protein and transport to the cell surface. In to in RIC-3 is not an for the surface of 5-HT3A in as it is not expressed to the in the or of receptor function is that RIC-3 a role in receptor by the of receptor a that is a for or a to with receptor to in RIC-3 identified in a in for of a in the acetylcholine receptor to the of a that is for to In this in RIC-3 found to this an essential role for RIC-3 in acetylcholine receptor by in to and but not in RIC-3 localized to cell as receptors in an essential role for RIC-3 in the on the of this role of RIC-3 to receptors an inhibitory of RIC-3 observed on the function of α4β2 and α3β4 and receptors, with on receptors. In to the for RIC-3 on transport the cell identified role for RIC-3 in the transport of receptors to the cell surface. a role in the of the binding Although this role the and a for of receptors to the cell the of this that surface in the of RIC-3 and that the on the cell surface. the for RIC-3 in the transport of and the of the inhibitory function of RIC-3 on α4β2 and α3β4 and receptors and the lack of of RIC-3 on or receptors we to the role of RIC-3 on and 5-HT3A receptor transport to the cell surface. in with for receptor function we found role for RIC-3 on the surface transport of receptors not In to the of RIC-3 on receptor we observed a of 5-HT3A receptor surface and is not but in the or of the 5-HT3A we observed in cell surface in the presence of to the of 5-HT3A to in is the of surface of a in the cell type has been when at levels is a in the transport of cell surface In with a role for RIC-3 this RIC-3 exhibits co-localization with the we not significant levels of RIC-3 at the cell surface by or cell surface to the observed an is that in the study, receptors. RIC-3 at the cell surface by receptors. this is not the for 5-HT3A receptors, in with the low transient interaction that can significant levels of surface RIC-3 not when with 5-HT3A. low level of 5-HT3A and RIC-3 is with a transient or low to and the interaction to the of of RIC-3 on 5-HT3A receptors. a to 5-HT3A receptor we observed that the in 5-HT3A receptor surface to the level of a by functional an in receptor levels when RIC-3 in is to of a of the 5-HT3A been and receptor is the receptor the is by RIC-3 not this with a role for RIC-3 in 5-HT3A receptor by a as receptor observed for or receptors to and to the cell surface to the observed for we the of surface to receptors, it that the in cell surface levels at low levels for 3A to of the of in receptor the of RIC-3 on both surface and 5-HT3A receptor the of RIC-3 the cell and the low level interaction with 5-HT3A receptors that RIC-3 that is a for protein and transport to the cell surface. In to in RIC-3 is not an for the surface of 5-HT3A in as it is not expressed to the in the or of receptor function is that RIC-3 a role in receptor by the of receptor a that is a for or a to with receptor to in for the of the RIC-3 and of for on