Src regulates constitutive internalization and rapid resensitization of a cholecystokinin 2 receptor splice variant

Celia Chao, Kirk L. Ives, Elizabeth Goluszko, Andrey A. Kolokoltsov, Robert A. Davey, Courtney Townsend, Mark Hellmich

Research output: Contribution to journalArticle

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Abstract

The third intracellular loop domain of G protein-coupled receptors regulates their desensitization, internalization, and resensitization. Colorectal and pancreatic cancers, but not the nonmalignant tissue, express a splice variant of the cholecystokinin 2 receptor (CCK2R) called CCK2i4svR that, because of intron 4 retention, contains an additional 69 amino acids within its third intracellular loop domain. This structural alteration is associated with agonist-independent activation of Src kinase (Olszewska-Pazdrak, B., Townsend, C. M., Jr., and Hellmich, M. R. (2004) J. Biol. Chem. 279, 40400-40404). The purpose of the study was to determine the roles of intron 4 retention and Src kinase on CCK2i4svR desensitization, internalization, and resensitization. Gastrin1-17 (G17) binds to both CCK2R and CCK2i4svR and induces intracellular Ca2+ ([Ca2+]i) increases. Agonist-induced increases in [Ca2+]i were used to assess receptor activity. Src kinase activity was inhibited by transducing cells with a retrovirus containing a dominant-negative mutant Src (A430V). The subcellular location of enhanced green fluorescent protein-tagged receptors was monitored using laser scanning confocal microscopy. Both receptor variants desensitized at the same rate; however, CCK2i4svR resensitized five times faster than CCK2R. Without agonist, 80% of CCK2i4svR is located in an intracellular compartment. In contrast, 80% of CCK2R was located on the plasma membrane. Treatment with inverse agonist (YM022) or expression of dominant-negative Src blocked the constitutive internalization of CCK2i4svR, resulting in its accumulation on the plasma membrane. Expression of dominant-negative Src slowed the rate of CCK2i4svR resensitization. Inhibition of Src did not affect G17-induced internalization of either receptor variant. Constitutive internalization of CCK2i4svR increases its rate of resensitization by creating an intracellular pool of receptors that can rapidly recycle back to the plasma membrane.

Original languageEnglish (US)
Pages (from-to)33368-33373
Number of pages6
JournalJournal of Biological Chemistry
Volume280
Issue number39
DOIs
StatePublished - Sep 30 2005

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Cholecystokinin B Receptor
src-Family Kinases
Cell membranes
Cell Membrane
Introns
Confocal microscopy
Retroviridae
G-Protein-Coupled Receptors
Pancreatic Neoplasms
Confocal Microscopy
Colorectal Neoplasms
Chemical activation
Tissue
Scanning
Amino Acids
Lasers

ASJC Scopus subject areas

  • Biochemistry

Cite this

Src regulates constitutive internalization and rapid resensitization of a cholecystokinin 2 receptor splice variant. / Chao, Celia; Ives, Kirk L.; Goluszko, Elizabeth; Kolokoltsov, Andrey A.; Davey, Robert A.; Townsend, Courtney; Hellmich, Mark.

In: Journal of Biological Chemistry, Vol. 280, No. 39, 30.09.2005, p. 33368-33373.

Research output: Contribution to journalArticle

Chao, Celia ; Ives, Kirk L. ; Goluszko, Elizabeth ; Kolokoltsov, Andrey A. ; Davey, Robert A. ; Townsend, Courtney ; Hellmich, Mark. / Src regulates constitutive internalization and rapid resensitization of a cholecystokinin 2 receptor splice variant. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 39. pp. 33368-33373.
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abstract = "The third intracellular loop domain of G protein-coupled receptors regulates their desensitization, internalization, and resensitization. Colorectal and pancreatic cancers, but not the nonmalignant tissue, express a splice variant of the cholecystokinin 2 receptor (CCK2R) called CCK2i4svR that, because of intron 4 retention, contains an additional 69 amino acids within its third intracellular loop domain. This structural alteration is associated with agonist-independent activation of Src kinase (Olszewska-Pazdrak, B., Townsend, C. M., Jr., and Hellmich, M. R. (2004) J. Biol. Chem. 279, 40400-40404). The purpose of the study was to determine the roles of intron 4 retention and Src kinase on CCK2i4svR desensitization, internalization, and resensitization. Gastrin1-17 (G17) binds to both CCK2R and CCK2i4svR and induces intracellular Ca2+ ([Ca2+]i) increases. Agonist-induced increases in [Ca2+]i were used to assess receptor activity. Src kinase activity was inhibited by transducing cells with a retrovirus containing a dominant-negative mutant Src (A430V). The subcellular location of enhanced green fluorescent protein-tagged receptors was monitored using laser scanning confocal microscopy. Both receptor variants desensitized at the same rate; however, CCK2i4svR resensitized five times faster than CCK2R. Without agonist, 80{\%} of CCK2i4svR is located in an intracellular compartment. In contrast, 80{\%} of CCK2R was located on the plasma membrane. Treatment with inverse agonist (YM022) or expression of dominant-negative Src blocked the constitutive internalization of CCK2i4svR, resulting in its accumulation on the plasma membrane. Expression of dominant-negative Src slowed the rate of CCK2i4svR resensitization. Inhibition of Src did not affect G17-induced internalization of either receptor variant. Constitutive internalization of CCK2i4svR increases its rate of resensitization by creating an intracellular pool of receptors that can rapidly recycle back to the plasma membrane.",
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AU - Davey, Robert A.

AU - Townsend, Courtney

AU - Hellmich, Mark

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AB - The third intracellular loop domain of G protein-coupled receptors regulates their desensitization, internalization, and resensitization. Colorectal and pancreatic cancers, but not the nonmalignant tissue, express a splice variant of the cholecystokinin 2 receptor (CCK2R) called CCK2i4svR that, because of intron 4 retention, contains an additional 69 amino acids within its third intracellular loop domain. This structural alteration is associated with agonist-independent activation of Src kinase (Olszewska-Pazdrak, B., Townsend, C. M., Jr., and Hellmich, M. R. (2004) J. Biol. Chem. 279, 40400-40404). The purpose of the study was to determine the roles of intron 4 retention and Src kinase on CCK2i4svR desensitization, internalization, and resensitization. Gastrin1-17 (G17) binds to both CCK2R and CCK2i4svR and induces intracellular Ca2+ ([Ca2+]i) increases. Agonist-induced increases in [Ca2+]i were used to assess receptor activity. Src kinase activity was inhibited by transducing cells with a retrovirus containing a dominant-negative mutant Src (A430V). The subcellular location of enhanced green fluorescent protein-tagged receptors was monitored using laser scanning confocal microscopy. Both receptor variants desensitized at the same rate; however, CCK2i4svR resensitized five times faster than CCK2R. Without agonist, 80% of CCK2i4svR is located in an intracellular compartment. In contrast, 80% of CCK2R was located on the plasma membrane. Treatment with inverse agonist (YM022) or expression of dominant-negative Src blocked the constitutive internalization of CCK2i4svR, resulting in its accumulation on the plasma membrane. Expression of dominant-negative Src slowed the rate of CCK2i4svR resensitization. Inhibition of Src did not affect G17-induced internalization of either receptor variant. Constitutive internalization of CCK2i4svR increases its rate of resensitization by creating an intracellular pool of receptors that can rapidly recycle back to the plasma membrane.

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