Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888

Autores
Young, Steven H.; Rey, Osvaldo; Sinnett Smith, James; Rozengurt, Enrique
Año de publicación
2014
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
To clarify the mechanism(s) underlying intracellular Ca2+ concentration ([Ca2+]i) oscillations induced by an elevation in extracellular Ca2+ concentration ([Ca2+]e) via the extracellular Ca2+-sensing receptor (CaR), we analyzed the pattern of [Ca2+]i response in multiple (2,303) individual HEK-293 cells transfected with the human CaR. An increase in the [Ca2+]e from 1.5 to 3 mM produced oscillatory fluctuations in [Ca2+]i in 70% of the cell population. To determine the role of PKC in the generation of [Ca2+]i oscillations, cells were exposed to increasing concentrations (0.5–5 μM) of the preferential PKC inhibitor Ro-31-8220 before stimulation by extracellular Ca2+. Ro-31-8220 at 3–5 μM completely eliminated the [Ca2+]e-evoked [Ca2+]i oscillations and transformed the pattern to a peak and sustained plateau response. Treatment with other broad PKC inhibitors, including GFI or Gö6983, produced an identical response. Similarly, treatment with Ro-31-8220 or GFI eliminated [Ca2+]e-evoked [Ca2+]i oscillations in colon-derived SW-480 cells expressing the CaR. Treatment with inhibitors targeting classic PKCs, including Gö6976 and Ro-32-0432 as well as small interfering RNA-mediated knockdown of PKCα, strikingly reduced the proportion of cell displaying [Ca2+]e-evoked [Ca2+]i oscillations. Furthermore, none of the cells analyzed expressing a CaR mutant in which the major PKC phosphorylation site Thr888 was converted to alanine (CaRT888A) showed [Ca2+]i oscillations after CaR activation. Our results show that [Ca2+]i oscillations induced by activation of the CaR in response to an increase in extracellular Ca2+ or exposure to the calcimimetic R-568 result from negative feedback involving PKCα-mediated phosphorylation of the CaR at Thr888.
Fil: Young, Steven H.. University of California at Los Angeles; Estados Unidos
Fil: Rey, Osvaldo. University of California at Los Angeles; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina
Fil: Sinnett Smith, James. University of California at Los Angeles; Estados Unidos
Fil: Rozengurt, Enrique. University of California at Los Angeles; Estados Unidos
Materia
Casr
Gpcr
Proliferation
Signal Transduction
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/18639

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spelling Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888Young, Steven H.Rey, OsvaldoSinnett Smith, JamesRozengurt, EnriqueCasrGpcrProliferationSignal Transductionhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1To clarify the mechanism(s) underlying intracellular Ca2+ concentration ([Ca2+]i) oscillations induced by an elevation in extracellular Ca2+ concentration ([Ca2+]e) via the extracellular Ca2+-sensing receptor (CaR), we analyzed the pattern of [Ca2+]i response in multiple (2,303) individual HEK-293 cells transfected with the human CaR. An increase in the [Ca2+]e from 1.5 to 3 mM produced oscillatory fluctuations in [Ca2+]i in 70% of the cell population. To determine the role of PKC in the generation of [Ca2+]i oscillations, cells were exposed to increasing concentrations (0.5–5 μM) of the preferential PKC inhibitor Ro-31-8220 before stimulation by extracellular Ca2+. Ro-31-8220 at 3–5 μM completely eliminated the [Ca2+]e-evoked [Ca2+]i oscillations and transformed the pattern to a peak and sustained plateau response. Treatment with other broad PKC inhibitors, including GFI or Gö6983, produced an identical response. Similarly, treatment with Ro-31-8220 or GFI eliminated [Ca2+]e-evoked [Ca2+]i oscillations in colon-derived SW-480 cells expressing the CaR. Treatment with inhibitors targeting classic PKCs, including Gö6976 and Ro-32-0432 as well as small interfering RNA-mediated knockdown of PKCα, strikingly reduced the proportion of cell displaying [Ca2+]e-evoked [Ca2+]i oscillations. Furthermore, none of the cells analyzed expressing a CaR mutant in which the major PKC phosphorylation site Thr888 was converted to alanine (CaRT888A) showed [Ca2+]i oscillations after CaR activation. Our results show that [Ca2+]i oscillations induced by activation of the CaR in response to an increase in extracellular Ca2+ or exposure to the calcimimetic R-568 result from negative feedback involving PKCα-mediated phosphorylation of the CaR at Thr888.Fil: Young, Steven H.. University of California at Los Angeles; Estados UnidosFil: Rey, Osvaldo. University of California at Los Angeles; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; ArgentinaFil: Sinnett Smith, James. University of California at Los Angeles; Estados UnidosFil: Rozengurt, Enrique. University of California at Los Angeles; Estados UnidosAmerican Physiological Society2014-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/18639Young, Steven H.; Rey, Osvaldo; Sinnett Smith, James; Rozengurt, Enrique; Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888; American Physiological Society; American Journal of Physiology-cell Physiology; 306; 3; 3-2014; 298-3060363-61431522-1563CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/http://ajpcell.physiology.org/content/306/3/C298.longinfo:eu-repo/semantics/altIdentifier/doi/10.1152/ajpcell.00194.2013info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:54:02Zoai:ri.conicet.gov.ar:11336/18639instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 09:54:02.472CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888
title Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888
spellingShingle Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888
Young, Steven H.
Casr
Gpcr
Proliferation
Signal Transduction
title_short Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888
title_full Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888
title_fullStr Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888
title_full_unstemmed Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888
title_sort Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888
dc.creator.none.fl_str_mv Young, Steven H.
Rey, Osvaldo
Sinnett Smith, James
Rozengurt, Enrique
author Young, Steven H.
author_facet Young, Steven H.
Rey, Osvaldo
Sinnett Smith, James
Rozengurt, Enrique
author_role author
author2 Rey, Osvaldo
Sinnett Smith, James
Rozengurt, Enrique
author2_role author
author
author
dc.subject.none.fl_str_mv Casr
Gpcr
Proliferation
Signal Transduction
topic Casr
Gpcr
Proliferation
Signal Transduction
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv To clarify the mechanism(s) underlying intracellular Ca2+ concentration ([Ca2+]i) oscillations induced by an elevation in extracellular Ca2+ concentration ([Ca2+]e) via the extracellular Ca2+-sensing receptor (CaR), we analyzed the pattern of [Ca2+]i response in multiple (2,303) individual HEK-293 cells transfected with the human CaR. An increase in the [Ca2+]e from 1.5 to 3 mM produced oscillatory fluctuations in [Ca2+]i in 70% of the cell population. To determine the role of PKC in the generation of [Ca2+]i oscillations, cells were exposed to increasing concentrations (0.5–5 μM) of the preferential PKC inhibitor Ro-31-8220 before stimulation by extracellular Ca2+. Ro-31-8220 at 3–5 μM completely eliminated the [Ca2+]e-evoked [Ca2+]i oscillations and transformed the pattern to a peak and sustained plateau response. Treatment with other broad PKC inhibitors, including GFI or Gö6983, produced an identical response. Similarly, treatment with Ro-31-8220 or GFI eliminated [Ca2+]e-evoked [Ca2+]i oscillations in colon-derived SW-480 cells expressing the CaR. Treatment with inhibitors targeting classic PKCs, including Gö6976 and Ro-32-0432 as well as small interfering RNA-mediated knockdown of PKCα, strikingly reduced the proportion of cell displaying [Ca2+]e-evoked [Ca2+]i oscillations. Furthermore, none of the cells analyzed expressing a CaR mutant in which the major PKC phosphorylation site Thr888 was converted to alanine (CaRT888A) showed [Ca2+]i oscillations after CaR activation. Our results show that [Ca2+]i oscillations induced by activation of the CaR in response to an increase in extracellular Ca2+ or exposure to the calcimimetic R-568 result from negative feedback involving PKCα-mediated phosphorylation of the CaR at Thr888.
Fil: Young, Steven H.. University of California at Los Angeles; Estados Unidos
Fil: Rey, Osvaldo. University of California at Los Angeles; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina
Fil: Sinnett Smith, James. University of California at Los Angeles; Estados Unidos
Fil: Rozengurt, Enrique. University of California at Los Angeles; Estados Unidos
description To clarify the mechanism(s) underlying intracellular Ca2+ concentration ([Ca2+]i) oscillations induced by an elevation in extracellular Ca2+ concentration ([Ca2+]e) via the extracellular Ca2+-sensing receptor (CaR), we analyzed the pattern of [Ca2+]i response in multiple (2,303) individual HEK-293 cells transfected with the human CaR. An increase in the [Ca2+]e from 1.5 to 3 mM produced oscillatory fluctuations in [Ca2+]i in 70% of the cell population. To determine the role of PKC in the generation of [Ca2+]i oscillations, cells were exposed to increasing concentrations (0.5–5 μM) of the preferential PKC inhibitor Ro-31-8220 before stimulation by extracellular Ca2+. Ro-31-8220 at 3–5 μM completely eliminated the [Ca2+]e-evoked [Ca2+]i oscillations and transformed the pattern to a peak and sustained plateau response. Treatment with other broad PKC inhibitors, including GFI or Gö6983, produced an identical response. Similarly, treatment with Ro-31-8220 or GFI eliminated [Ca2+]e-evoked [Ca2+]i oscillations in colon-derived SW-480 cells expressing the CaR. Treatment with inhibitors targeting classic PKCs, including Gö6976 and Ro-32-0432 as well as small interfering RNA-mediated knockdown of PKCα, strikingly reduced the proportion of cell displaying [Ca2+]e-evoked [Ca2+]i oscillations. Furthermore, none of the cells analyzed expressing a CaR mutant in which the major PKC phosphorylation site Thr888 was converted to alanine (CaRT888A) showed [Ca2+]i oscillations after CaR activation. Our results show that [Ca2+]i oscillations induced by activation of the CaR in response to an increase in extracellular Ca2+ or exposure to the calcimimetic R-568 result from negative feedback involving PKCα-mediated phosphorylation of the CaR at Thr888.
publishDate 2014
dc.date.none.fl_str_mv 2014-03
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/18639
Young, Steven H.; Rey, Osvaldo; Sinnett Smith, James; Rozengurt, Enrique; Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888; American Physiological Society; American Journal of Physiology-cell Physiology; 306; 3; 3-2014; 298-306
0363-6143
1522-1563
CONICET Digital
CONICET
url http://hdl.handle.net/11336/18639
identifier_str_mv Young, Steven H.; Rey, Osvaldo; Sinnett Smith, James; Rozengurt, Enrique; Intracellular Ca2+ oscillations generated via the Ca2+-sensing receptor are mediated by negative feedback by PKCα at Thr888; American Physiological Society; American Journal of Physiology-cell Physiology; 306; 3; 3-2014; 298-306
0363-6143
1522-1563
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://ajpcell.physiology.org/content/306/3/C298.long
info:eu-repo/semantics/altIdentifier/doi/10.1152/ajpcell.00194.2013
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv American Physiological Society
publisher.none.fl_str_mv American Physiological Society
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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