The C-terminal tail of CRTH2 is a key molecular determinant that constrains GalphaI- and downstream-signaling cascade activation

Ralf Schroeder; Nicole Merten; Jesper Mosolff Mathiesen; Lene Martini; Anamarija Kruljac Letunic; Friederike Krop; Andree Blaukat; Ye Fang; Elizabeth Tran; Trond Ulven; Christel Drewke; Jennifer Whistler; Leonardo Pardo; Jesus Gomeza; Evi Kostenis

doi:10.1074/jbc.M806867200

The C-terminal tail of CRTH2 is a key molecular determinant that constrains GalphaI- and downstream-signaling cascade activation

Ralf Schroeder, Nicole Merten, Jesper Mosolff Mathiesen, Lene Martini, Anamarija Kruljac Letunic, Friederike Krop, Andree Blaukat, Ye Fang, Elizabeth Tran, Trond Ulven, Christel Drewke, Jennifer Whistler, Leonardo Pardo, Jesus Gomeza, Evi Kostenis

51 Citations (Scopus)

Abstract

Prostaglandin D(2) activation of the seven transmembrane receptor CRTH2 regulates numerous cell functions that are important in inflammatory diseases such as asthma. Despite its disease implication, no studies to date aimed at identifying receptor domains governing signaling and surface expression of human CRTH2. We tested the hypothesis that CRTH2 may take advantage of its C-tail to silence its own signaling and that this mechanism may explain the poor functional responses observed with CRTH2 in heterologous expression systems. Although the C terminus is a critical determinant for retention of CRTH2 at the plasma membrane, presence of this domain confers a signaling-compromised conformation onto the receptor. Indeed, a mutant receptor lacking the major portion of its C-terminal tail displays paradoxically enhanced Galphai and ERK1/2 activation in spite of enhanced constitutive and agonist-mediated internalization. Enhanced activation of Galphai proteins and downstream signaling cascades is likely due to the inability of the tail-truncated receptor to recruit beta-arrestin2 and undergo homologous desensitization. Unexpectedly, CRTH2 is not phosphorylated upon agonist-stimulation, a primary mechanism by which GPCR activity is regulated. Dynamic mass redistribution assays, which allow label-free monitoring of all major G protein pathways in real time, confirm that the C terminus inhibits Galphai-signaling of CRTH2 but does not encode G protein specificity determinants. We propose that intrinsic CRTH2 inhibition by its C terminus may represent a rather unappreciated strategy employed by a GPCR to specify the extent of G protein activation and that this mechanism may compensate for the absence of the classical phosphorylation-dependent signal attenuation.

Original language	English
Journal	Journal of Biological Chemistry
Volume	284
Issue number	2
Pages (from-to)	1324-1336
Number of pages	13
ISSN	0021-9258
DOIs	https://doi.org/10.1074/jbc.M806867200
Publication status	Published - 2009

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Schroeder, R., Merten, N., Mathiesen, J. M., Martini, L., Letunic, A. K., Krop, F., Blaukat, A., Fang, Y., Tran, E., Ulven, T., Drewke, C., Whistler, J., Pardo, L., Gomeza, J., & Kostenis, E. (2009). The C-terminal tail of CRTH2 is a key molecular determinant that constrains GalphaI- and downstream-signaling cascade activation. Journal of Biological Chemistry, 284(2), 1324-1336. https://doi.org/10.1074/jbc.M806867200

Schroeder, R, Merten, N, Mathiesen, JM, Martini, L, Letunic, AK, Krop, F, Blaukat, A, Fang, Y, Tran, E, Ulven, T, Drewke, C, Whistler, J, Pardo, L, Gomeza, J & Kostenis, E 2009, 'The C-terminal tail of CRTH2 is a key molecular determinant that constrains GalphaI- and downstream-signaling cascade activation', Journal of Biological Chemistry, vol. 284, no. 2, pp. 1324-1336. https://doi.org/10.1074/jbc.M806867200

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title = "The C-terminal tail of CRTH2 is a key molecular determinant that constrains GalphaI- and downstream-signaling cascade activation",

abstract = "Prostaglandin D(2) activation of the seven transmembrane receptor CRTH2 regulates numerous cell functions that are important in inflammatory diseases such as asthma. Despite its disease implication, no studies to date aimed at identifying receptor domains governing signaling and surface expression of human CRTH2. We tested the hypothesis that CRTH2 may take advantage of its C-tail to silence its own signaling and that this mechanism may explain the poor functional responses observed with CRTH2 in heterologous expression systems. Although the C terminus is a critical determinant for retention of CRTH2 at the plasma membrane, presence of this domain confers a signaling-compromised conformation onto the receptor. Indeed, a mutant receptor lacking the major portion of its C-terminal tail displays paradoxically enhanced Galphai and ERK1/2 activation in spite of enhanced constitutive and agonist-mediated internalization. Enhanced activation of Galphai proteins and downstream signaling cascades is likely due to the inability of the tail-truncated receptor to recruit beta-arrestin2 and undergo homologous desensitization. Unexpectedly, CRTH2 is not phosphorylated upon agonist-stimulation, a primary mechanism by which GPCR activity is regulated. Dynamic mass redistribution assays, which allow label-free monitoring of all major G protein pathways in real time, confirm that the C terminus inhibits Galphai-signaling of CRTH2 but does not encode G protein specificity determinants. We propose that intrinsic CRTH2 inhibition by its C terminus may represent a rather unappreciated strategy employed by a GPCR to specify the extent of G protein activation and that this mechanism may compensate for the absence of the classical phosphorylation-dependent signal attenuation.",

author = "Ralf Schroeder and Nicole Merten and Mathiesen, {Jesper Mosolff} and Lene Martini and Letunic, {Anamarija Kruljac} and Friederike Krop and Andree Blaukat and Ye Fang and Elizabeth Tran and Trond Ulven and Christel Drewke and Jennifer Whistler and Leonardo Pardo and Jesus Gomeza and Evi Kostenis",

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doi = "10.1074/jbc.M806867200",

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T1 - The C-terminal tail of CRTH2 is a key molecular determinant that constrains GalphaI- and downstream-signaling cascade activation

AU - Schroeder, Ralf

AU - Merten, Nicole

AU - Mathiesen, Jesper Mosolff

AU - Martini, Lene

AU - Letunic, Anamarija Kruljac

AU - Krop, Friederike

AU - Blaukat, Andree

AU - Fang, Ye

AU - Tran, Elizabeth

AU - Ulven, Trond

AU - Drewke, Christel

AU - Whistler, Jennifer

AU - Pardo, Leonardo

AU - Gomeza, Jesus

AU - Kostenis, Evi

PY - 2009

Y1 - 2009

N2 - Prostaglandin D(2) activation of the seven transmembrane receptor CRTH2 regulates numerous cell functions that are important in inflammatory diseases such as asthma. Despite its disease implication, no studies to date aimed at identifying receptor domains governing signaling and surface expression of human CRTH2. We tested the hypothesis that CRTH2 may take advantage of its C-tail to silence its own signaling and that this mechanism may explain the poor functional responses observed with CRTH2 in heterologous expression systems. Although the C terminus is a critical determinant for retention of CRTH2 at the plasma membrane, presence of this domain confers a signaling-compromised conformation onto the receptor. Indeed, a mutant receptor lacking the major portion of its C-terminal tail displays paradoxically enhanced Galphai and ERK1/2 activation in spite of enhanced constitutive and agonist-mediated internalization. Enhanced activation of Galphai proteins and downstream signaling cascades is likely due to the inability of the tail-truncated receptor to recruit beta-arrestin2 and undergo homologous desensitization. Unexpectedly, CRTH2 is not phosphorylated upon agonist-stimulation, a primary mechanism by which GPCR activity is regulated. Dynamic mass redistribution assays, which allow label-free monitoring of all major G protein pathways in real time, confirm that the C terminus inhibits Galphai-signaling of CRTH2 but does not encode G protein specificity determinants. We propose that intrinsic CRTH2 inhibition by its C terminus may represent a rather unappreciated strategy employed by a GPCR to specify the extent of G protein activation and that this mechanism may compensate for the absence of the classical phosphorylation-dependent signal attenuation.

AB - Prostaglandin D(2) activation of the seven transmembrane receptor CRTH2 regulates numerous cell functions that are important in inflammatory diseases such as asthma. Despite its disease implication, no studies to date aimed at identifying receptor domains governing signaling and surface expression of human CRTH2. We tested the hypothesis that CRTH2 may take advantage of its C-tail to silence its own signaling and that this mechanism may explain the poor functional responses observed with CRTH2 in heterologous expression systems. Although the C terminus is a critical determinant for retention of CRTH2 at the plasma membrane, presence of this domain confers a signaling-compromised conformation onto the receptor. Indeed, a mutant receptor lacking the major portion of its C-terminal tail displays paradoxically enhanced Galphai and ERK1/2 activation in spite of enhanced constitutive and agonist-mediated internalization. Enhanced activation of Galphai proteins and downstream signaling cascades is likely due to the inability of the tail-truncated receptor to recruit beta-arrestin2 and undergo homologous desensitization. Unexpectedly, CRTH2 is not phosphorylated upon agonist-stimulation, a primary mechanism by which GPCR activity is regulated. Dynamic mass redistribution assays, which allow label-free monitoring of all major G protein pathways in real time, confirm that the C terminus inhibits Galphai-signaling of CRTH2 but does not encode G protein specificity determinants. We propose that intrinsic CRTH2 inhibition by its C terminus may represent a rather unappreciated strategy employed by a GPCR to specify the extent of G protein activation and that this mechanism may compensate for the absence of the classical phosphorylation-dependent signal attenuation.

U2 - 10.1074/jbc.M806867200

DO - 10.1074/jbc.M806867200

M3 - Journal article

SN - 0021-9258

VL - 284

SP - 1324

EP - 1336

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 2

ER -

The C-terminal tail of CRTH2 is a key molecular determinant that constrains GalphaI- and downstream-signaling cascade activation

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