Role of the Differentially Spliced Carboxyl Terminus in Thromboxane A2 Receptor Trafficking

The thromboxane A2 receptor (TP) is a G protein-coupled receptor that is expressed as two alternatively spliced isoforms, α (343 residues) and β (407 residues) that share the first 328 residues. We have previously shown that TPβ, but not TPα, undergoes agonist-induced internalization in a dynamin-, GRK-, and arrestin-dependent manner. In the present report, we demonstrate that TPβ, but not TPα, also undergoes tonic internalization. Tonic internalization of TPβ was temperature- and dynamin-dependent and was inhibited by sucrose and NH4Cl treatment but unaffected by wild-type or dominant-negative GRKs or arrestins. Truncation and site-directed mutagenesis revealed that a YX 3φ motif (whereX is any residue and φ is a bulky hydrophobic residue) found in the proximal portion of the carboxyl-terminal tail of TPβ was critical for tonic internalization but had no role in agonist-induced internalization. Interestingly, introduction of either a YX 2φ or YX 3φ motif in the carboxyl-terminal tail of TPα induced tonic internalization of this receptor. Additional analysis revealed that tonically internalized TPβ undergoes recycling back to the cell surface suggesting that tonic internalization may play a role in maintaining an intracellular pool of TPβ. Our data demonstrate the presence of distinct signals for tonic and agonist-induced internalization of TPβ and represent the first report of a YX 3φ motif involved in tonic internalization of a cell surface receptor. The thromboxane A2 receptor (TP) is a G protein-coupled receptor that is expressed as two alternatively spliced isoforms, α (343 residues) and β (407 residues) that share the first 328 residues. We have previously shown that TPβ, but not TPα, undergoes agonist-induced internalization in a dynamin-, GRK-, and arrestin-dependent manner. In the present report, we demonstrate that TPβ, but not TPα, also undergoes tonic internalization. Tonic internalization of TPβ was temperature- and dynamin-dependent and was inhibited by sucrose and NH4Cl treatment but unaffected by wild-type or dominant-negative GRKs or arrestins. Truncation and site-directed mutagenesis revealed that a YX 3φ motif (whereX is any residue and φ is a bulky hydrophobic residue) found in the proximal portion of the carboxyl-terminal tail of TPβ was critical for tonic internalization but had no role in agonist-induced internalization. Interestingly, introduction of either a YX 2φ or YX 3φ motif in the carboxyl-terminal tail of TPα induced tonic internalization of this receptor. Additional analysis revealed that tonically internalized TPβ undergoes recycling back to the cell surface suggesting that tonic internalization may play a role in maintaining an intracellular pool of TPβ. Our data demonstrate the presence of distinct signals for tonic and agonist-induced internalization of TPβ and represent the first report of a YX 3φ motif involved in tonic internalization of a cell surface receptor. G protein-coupled receptor thromboxane A2 receptor G protein-coupled receptor kinase phosphate-buffered saline enzyme-linked immunosorbent assay human embryonic kidney Dulbecco's modified Eagle's medium bovine serum albumin Tris-buffered saline hemagglutinin polymerase chain reaction Cell surface receptors provide a primary mechanism by which cells perceive their environment. Many cell surface receptors are dynamically regulated and often undergo a process of endocytic sorting (1Mukherjee S. Ghosh R.N. Maxfield F.R. Physiol. Rev. receptors G protein-coupled and sorting is often by for and the receptors undergo or tonic internalization and have that the and receptors also undergo tonic internalization Cell no motif for tonic internalization of and and have shown to for a of receptor (1Mukherjee S. Ghosh R.N. Maxfield F.R. Physiol. Rev. have and the chain of the Cell and and the of to in a of and kidney Cell Physiol. is by the of by and thromboxane of a of cell and cells Physiol. is a of and cell and a The thromboxane (TP) is by a that is alternatively spliced in the in two TPα (343 residues) and TPβ (407 residues) that share the first 328 S. S. a we that TPβ, but not TPα, undergoes agonist-induced internalization in a of cell of TPβ was dynamin-, GRK-, and arrestin-dependent in suggesting the of receptor and in this Additional of the role of in this process revealed that agonist-induced internalization of TPα and TPβ but not of a residue of carboxyl-terminal revealed that a and in TPβ was for internalization. the of we that TPβ, but not TPα, also undergoes tonic internalization. In the present we the involved in tonic internalization of TPβ. that a YX 3φ motif found in the proximal portion of the carboxyl-terminal tail of TPβ is for tonic of the of the two of the human thromboxane A2 receptor is shown in tonic internalization of TPα and TPβ, receptors expressed in have shown that the of TPα and TPβ are S. of a the of the receptors not the of the receptors as by their for of not analysis of agonist-induced internalization of thromboxane A2 we that TPβ undergo tonic internalization this we a of cells the for and that tonic receptor internalization shown in is of TPβ to intracellular internalization of TPα is of intracellular and cell revealed that of TPβ was tonically to a a that TPβ, but not TPα, undergoes tonic internalization. In the cells are for or TPα and TPβ the cell Interestingly, the of TPβ to undergo tonic internalization is a agonist-induced internalization of TPβ not and the but distinct tonic internalization of the receptor which not analysis of α and β in receptors in the to any treatment to receptors that present the cell was as receptor in cells TPα and TPβ a a TPβ, to for Tonic internalization of TPβ in the presence or of the receptor the of NH4Cl and or was by and represent the of intracellular to of was the shown represent the of of cells was for to for that the internalization of TPβ was not by of thromboxane or by of in the cells the or the for to had no internalization of TPβ that the receptor is tonic internalization The for TPα and TPβ in and which and and which as as in cells of thromboxane receptors not demonstrate that the tonic internalization of TPα and TPβ is a of the previously that agonist-induced internalization of TPβ is dynamin-, and arrestin-dependent we the role of in tonic internalization of TPβ. analysis of TPβ in the presence of dominant-negative of Cell and was of inhibited tonic internalization and had no not Interestingly, of but not or also in an cell surface of TPβ as by not In had no cell surface of TPα not cells of as sucrose and tonic internalization of TPβ was also demonstrate that tonic and agonist-induced internalization of TPβ are tonically internalized TPβ is to a mechanism of GRKs and the of TPβ critical in tonic internalization of the we this to any residues. first to this in cells that of receptor The of to the to have no tonic internalization agonist-induced internalization was of an tonic internalization the found and to play a critical role in tonic internalization of TPβ. of this of TPβ the of TPα that in TPβ an of a tonic internalization role for internalization in tonic of a of receptors (1Mukherjee S. Ghosh R.N. Maxfield F.R. Physiol. Rev. Cell Rev. Cell this we a TPβ and tonic and agonist-induced internalization. not undergo any tonic internalization suggesting a critical role for in this Additional and in TPβ to in an to a motif for tonic internalization. The and of TPβ to undergo tonic was inhibited of agonist-induced internalization of TPβ suggesting that the motif for tonic internalization is distinct the for agonist-induced of TPβ data demonstrate that the motif a critical role in tonic internalization of TPβ. is to the motif as an role in tonic internalization of the receptor and receptor Rev. Cell internalization of tail of cells of TPβ receptor of for the to for tonic internalization of receptor was by of tonic internalization of the receptor was as in of the involved in tonic internalization of and to analysis of the tonic internalization of receptor of intracellular as in internalization of the receptor cells receptors and the of receptors the cell surface of was by as The represent the of in the of in tonic we and in the of Interestingly, introduction of a in TPα, a induced tonic internalization of TPα of to in the inhibited tonic the of the hydrophobic residue in this process In an to the of the the and hydrophobic we a and in the to a motif to that found in TPβ. not the internalization induced by the residue in that YX 2φ and YX 3φ as internalization Interestingly, a a YX 3φ motif but residue to the in TPβ and TPα not tonic internalization of that the of the in the receptor tail is also an in this of agonist-induced internalization of TPα not Our data that the the and the hydrophobic residue and the of the YX motif in the receptor tail are in tonic internalization of the thromboxane receptor. is to that a found in the intracellular of receptor is not to tonic internalization this is not for of this motif in TPβ not tonic internalization not of receptors in cells introduction of a residue in the tail of cells of receptor of of for TPα of TPα was by intracellular was as in tonic internalization also for Cell is to the role of this that tonic internalization of the receptor an intracellular pool of receptors that is to the cell surface receptors a role to tonic internalization of TPβ, we that receptors back to the cell surface tonic internalization. this cell surface receptors and to undergo tonic internalization for The cells to the cell surface in a and receptor by TPβ was cell surface was was of the intracellular receptors to the cell surface recycling was not by or to of receptors intracellular receptors the of cell surface receptors data that is recycling of the tonically internalized TPβ the cell surface and an intracellular to for the receptor tonic internalization of TPβ to an intracellular pool of receptors that to the cell surface to internalized β receptors to the cell receptors in the and for to tonic internalization. the cell surface was of the two cells for to recycling of receptors to the cell surface for shown are of motif is of the involved in the of sorting signals to the motif have shown to the and of the and in The critical not to in the of and may by S. The the and the of to the the and the of cell surface is involved in the of to and also that is a that is in the of the The of this a sorting suggesting that is also involved in the and sorting of the for of sorting signals and of a the revealed that was no for the presence of any of the or φ the critical residue that not by any residue a in sorting Cell S. S. and for S. S. was shown that a for was also in not the YX 3φ motif found in TPβ a and a to of the that to the receptor a and a the and or are also found of the motif of the and to TPβ, the receptor and have a the receptor and have a the the for is also a in or the may for to cell the in a motif of and to and The of the motif the may also Cell of the motif in by a residue to the was to S. Cell to in the TPα also a was in the of hemagglutinin to an internalization In internalization was the of the to the cell that the of the was Rev. Cell a distinct motif in the of a G protein-coupled receptor for tonic internalization. the the and the hydrophobic residue and the of the YX 3φ motif in the receptor tail to in this In signals that in the YX 3φ motif may the of the receptor. analysis of and for the of the YX 3φ to two in the of TPβ, the YX 3φ motif and the and to tonic and agonist-induced analysis of the of their provide as to their role in receptor to YX 3φ and which to the and may in the of the receptor of the The YX 3φ motif for involved in tonic internalization of the receptor the to involved in agonist-induced demonstrate that the of the of the thromboxane A2 receptor that distinct We had previously shown that TPβ, but not TPα, undergo agonist-induced internalization In the present report, we demonstrate that TPβ is of tonic internalization. Tonic internalization was to a YX 3φ which is distinct the for and is the first motif for tonic internalization of a TPα and TPβ to in the of receptors as as in their to the and involved in Cell surface receptors provide a primary mechanism by which cells perceive their environment. Many cell surface receptors are dynamically regulated and often undergo a process of endocytic sorting (1Mukherjee S. Ghosh R.N. Maxfield F.R. Physiol. Rev. receptors G protein-coupled and sorting is often by for and the receptors undergo or tonic internalization and have that the and receptors also undergo tonic internalization Cell no motif for tonic internalization of and and have shown to for a of receptor (1Mukherjee S. Ghosh R.N. Maxfield F.R. Physiol. Rev. have and the chain of the Cell and and the of to in a of and kidney Cell Physiol. is by the of by and thromboxane of a of cell and cells Physiol. is a of and cell and a The thromboxane (TP) is by a that is alternatively spliced in the in two TPα (343 residues) and TPβ (407 residues) that share the first 328 S. S. In a we that TPβ, but not TPα, undergoes agonist-induced internalization in a of cell of TPβ was dynamin-, GRK-, and arrestin-dependent in suggesting the of receptor and in this Additional of the role of in this process revealed that agonist-induced internalization of TPα and TPβ but not of a residue of carboxyl-terminal revealed that a and in TPβ was for internalization. the of we that TPβ, but not TPα, also undergoes tonic internalization. In the present we the involved in tonic internalization of TPβ. that a YX 3φ motif found in the proximal portion of the carboxyl-terminal tail of TPβ is for tonic internalization. of the of the two of the human thromboxane A2 receptor is shown in tonic internalization of TPα and TPβ, receptors expressed in have shown that the of TPα and TPβ are S. of a the of the receptors not the of the receptors as by their for of not analysis of agonist-induced internalization of thromboxane A2 we that TPβ undergo tonic internalization this we a of cells the for and that tonic receptor internalization shown in is of TPβ to intracellular internalization of TPα is of intracellular and cell revealed that of TPβ was tonically to a a that TPβ, but not TPα, undergoes tonic internalization. In the cells are for or TPα and TPβ the cell Interestingly, the of TPβ to undergo tonic internalization is a agonist-induced internalization of TPβ not and the but distinct tonic internalization of the receptor which not that the internalization of TPβ was not by of thromboxane or by of in the cells the or the for to had no internalization of TPβ that the receptor is tonic internalization The for TPα and TPβ in and which and and which as as in cells of thromboxane receptors not demonstrate that the tonic internalization of TPα and TPβ is a of the previously that agonist-induced internalization of TPβ is dynamin-, and arrestin-dependent we the role of in tonic internalization of TPβ. analysis of TPβ in the presence of dominant-negative of Cell and was of inhibited tonic internalization and had no not Interestingly, of but not or also in an cell surface of TPβ as by not In had no cell surface of TPα not cells of as sucrose and tonic internalization of TPβ was also demonstrate that tonic and agonist-induced internalization of TPβ are tonically internalized TPβ is to a mechanism of GRKs and the of TPβ critical in tonic internalization of the we this to any residues. first to this in cells that of receptor The of to the to have no tonic internalization agonist-induced internalization was of an tonic internalization the found and to play a critical role in tonic internalization of TPβ. of this of TPβ the of TPα that in TPβ an of a tonic internalization role for internalization in tonic of a of receptors (1Mukherjee S. Ghosh R.N. Maxfield F.R. Physiol. Rev. Cell Rev. Cell this we a TPβ and tonic and agonist-induced internalization. not undergo any tonic internalization suggesting a critical role for in this Additional and in TPβ to in an to a motif for tonic internalization. The and of TPβ to undergo tonic was inhibited of agonist-induced internalization of TPβ suggesting that the motif for tonic internalization is distinct the for agonist-induced of TPβ data demonstrate that the motif a critical role in tonic internalization of TPβ. is to the motif as an role in tonic internalization of the receptor and receptor Rev. Cell internalization of tail of cells of TPβ receptor of for the to for tonic internalization of receptor was by of tonic internalization of the receptor was as in of the involved in tonic internalization of and to analysis of the tonic internalization of receptor of intracellular as in internalization of the receptor cells receptors and the of receptors the cell surface of was by as The represent the of in the of in tonic we and in the of Interestingly, introduction of a in TPα, a induced tonic internalization of TPα of to in the inhibited tonic the of the hydrophobic residue in this process In an to the of the the and hydrophobic we a and in the to a motif to that found in TPβ. not the internalization induced by the residue in that YX 2φ and YX 3φ as internalization Interestingly, a a YX 3φ motif but residue to the in TPβ and TPα not tonic internalization of that the of the in the receptor tail is also an in this of agonist-induced internalization of TPα not Our data that the the and the hydrophobic residue and the of the YX motif in the receptor tail are in tonic internalization of the thromboxane receptor. is to that a found in the intracellular of receptor is not to tonic internalization this is not for of this motif in TPβ not tonic internalization not of receptors in cells introduction of a residue in the tail of cells of receptor of of for TPα of TPα was by intracellular was as in tonic internalization also for Cell is to the role of this that tonic internalization of the receptor an intracellular pool of receptors that is to the cell surface receptors a role to tonic internalization of TPβ, we that receptors back to the cell surface tonic internalization. this cell surface receptors and to undergo tonic internalization for The cells to the cell surface in a and receptor by TPβ was cell surface was was of the intracellular receptors to the cell surface recycling was not by or to of receptors intracellular receptors the of cell surface receptors data that is recycling of the tonically internalized TPβ the cell surface and an intracellular to for the receptor tonic internalization of TPβ to an intracellular pool of receptors that to the cell surface to internalized β receptors to the cell receptors in the and for to tonic internalization. the cell surface was of the two cells for to recycling of receptors to the cell surface for shown are of motif is of the involved in the of sorting signals to the motif have shown to the and of the and in The critical not to in the of and may by S. The the and the of to the the and the of cell surface is involved in the of to and also that is a that is in the of the The of this a sorting suggesting that is also involved in the and sorting of the for of sorting signals and of a the revealed that was no for the presence of any of the or φ the critical residue that not by any residue a in sorting Cell S. S. and for S. S. was shown that a for was also in not the YX 3φ motif found in TPβ a and a to of the that to the receptor a and a the and or are also found of the motif of the and to TPβ, the receptor and have a the receptor and have a the the for is also a in or the may for to cell the in a motif of and to and The of the motif the may also Cell of the motif in by a residue to the was to S. Cell to in the TPα also a was in the of hemagglutinin to an internalization In internalization was the of the to the cell that the of the was Rev. Cell a distinct motif in the of a G protein-coupled receptor for tonic internalization. the the and the hydrophobic residue and the of the YX 3φ motif in the receptor tail to in this In signals that in the YX 3φ motif may the of the receptor. analysis of and for the of the YX 3φ to two in the of TPβ, the YX 3φ motif and the and to tonic and agonist-induced analysis of the of their provide as to their role in receptor to YX 3φ and which to the and may in the of the receptor of the The YX 3φ motif for involved in tonic internalization of the receptor the to involved in agonist-induced demonstrate that the of the of the thromboxane A2 receptor that distinct We had previously shown that TPβ, but not TPα, undergo agonist-induced internalization In the present report, we demonstrate that TPβ is of tonic internalization. Tonic internalization was to a YX 3φ which is distinct the for and is the first motif for tonic internalization of a TPα and TPβ to in the of receptors as as in their to the and involved in of the of the two of the human thromboxane A2 receptor is shown in tonic internalization of TPα and TPβ, receptors expressed in have shown that the of TPα and TPβ are S. of a the of the receptors not the of the receptors as by their for of not analysis of agonist-induced internalization of thromboxane A2 we that TPβ undergo tonic internalization this we a of cells the for and that tonic receptor internalization shown in is of TPβ to intracellular internalization of TPα is of intracellular and cell revealed that of TPβ was tonically to a a that TPβ, but not TPα, undergoes tonic internalization. In the cells are for or TPα and TPβ the cell Interestingly, the of TPβ to undergo tonic internalization is a agonist-induced internalization of TPβ not and the but distinct tonic internalization of the receptor which not that the internalization of TPβ was not by of thromboxane or by of in the cells the or the for to had no internalization of TPβ that the receptor is tonic internalization The for TPα and TPβ in and which and and which as as in cells of thromboxane receptors not demonstrate that the tonic internalization of TPα and TPβ is a of the receptor. We previously that agonist-induced internalization of TPβ is dynamin-, and arrestin-dependent we the role of in tonic internalization of TPβ. analysis of TPβ in the presence of dominant-negative of Cell and was of inhibited tonic internalization and had no not Interestingly, of but not or also in an cell surface of TPβ as by not In had no cell surface of TPα not cells of as sucrose and tonic internalization of TPβ was also demonstrate that tonic and agonist-induced internalization of TPβ are tonically internalized TPβ is to a mechanism of GRKs and arrestins. the of TPβ critical in tonic internalization of the we this to any residues. first to this in cells that of receptor The of to the to have no tonic internalization agonist-induced internalization was of an tonic internalization the found and to play a critical role in tonic internalization of TPβ. of this of TPβ the of TPα that in TPβ an of a tonic internalization role for internalization in tonic of a of receptors (1Mukherjee S. Ghosh R.N. Maxfield F.R. Physiol. Rev. Cell Rev. Cell this we a TPβ and tonic and agonist-induced internalization. not undergo any tonic internalization suggesting a critical role for in this Additional and in TPβ to in an to a motif for tonic internalization. The and of TPβ to undergo tonic was inhibited of agonist-induced internalization of TPβ suggesting that the motif for tonic internalization is distinct the for agonist-induced of TPβ data demonstrate that the motif a critical role in tonic internalization of TPβ. is to the motif as an role in tonic internalization of the receptor and receptor Rev. Cell the of in tonic we and in the of Interestingly, introduction of a in TPα, a induced tonic internalization of TPα of to in the inhibited tonic the of the hydrophobic residue in this process In an to the of the the and hydrophobic we a and in the to a motif to that found in TPβ. not the internalization induced by the residue in that YX 2φ and YX 3φ as internalization Interestingly, a a YX 3φ motif but residue to the in TPβ and TPα not tonic internalization of that the of the in the receptor tail is also an in this of agonist-induced internalization of TPα not Our data that the the and the hydrophobic residue and the of the YX motif in the receptor tail are in tonic internalization of the thromboxane receptor. is to that a found in the intracellular of receptor is not to tonic internalization this is not for of this motif in TPβ not tonic internalization not tonic internalization also for Cell is to the role of this that tonic internalization of the receptor an intracellular pool of receptors that is to the cell surface receptors a role to tonic internalization of TPβ, we that receptors back to the cell surface tonic internalization. this cell surface receptors and to undergo tonic internalization for The cells to the cell surface in a and receptor by TPβ was cell surface was was of the intracellular receptors to the cell surface recycling was not by or to of receptors intracellular receptors the of cell surface receptors data that is recycling of the tonically internalized TPβ the cell surface and an intracellular to for the receptor tonic internalization of TPβ to an intracellular pool of receptors that to the cell surface to The motif is of the involved in the of sorting signals to the motif have shown to the and of the and in The critical not to in the of and may by S. The the and the of to the the and the of cell surface is involved in the of to and also that is a that is in the of the The of this a sorting suggesting that is also involved in the and sorting of the for of sorting signals and of a the revealed that was no for the presence of any of the or φ the critical residue that not by any residue a in sorting Cell S. S. and for S. S. was shown that a for was also in not the YX 3φ motif found in TPβ a and a to of the that to the receptor a and a the and or are also found of the motif of the and to TPβ, the receptor and have a the receptor and have a the the for is also a in or the may for to cell the in a motif of and to and The of the motif the may also Cell of the motif in by a residue to the was to S. Cell to in the TPα also a was in the of hemagglutinin to an internalization In internalization was the of the to the cell that the of the was Rev. Cell Our a distinct motif in the of a G protein-coupled receptor for tonic internalization. the the and the hydrophobic residue and the of the YX 3φ motif in the receptor tail to in this In signals that in the YX 3φ motif may the of the receptor. analysis of and for the of the YX 3φ to two in the of TPβ, the YX 3φ motif and the and to tonic and agonist-induced analysis of the of their provide as to their role in receptor to YX 3φ and which to the and may in the of the receptor of the The YX 3φ motif for involved in tonic internalization of the receptor the to involved in agonist-induced internalization. In demonstrate that the of the of the thromboxane A2 receptor that distinct We had previously shown that TPβ, but not TPα, undergo agonist-induced internalization In the present report, we demonstrate that TPβ is of tonic internalization. Tonic internalization was to a YX 3φ which is distinct the for and is the first motif for tonic internalization of a TPα and TPβ to in the of receptors as as in their to the and involved in

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