Neurotensin receptor-1 inducible palmitoylation is required for efficient receptor-mediated mitogenic-signaling within structured membrane microdomains

Yasser Heakal, Matthew P. Woll, Todd Fox, Kelly Seaton, Robert Levenson, Mark Kester

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Neurotensin receptor-1 (NTSR-1) is a G-protein coupled receptor (GPCR) that has been recently identified as a mediator of cancer progression. NTSR-1 and its endogenous ligand, neurotensin (NTS), are co-expressed in several breast cancer cell lines and breast cancer tumor samples. Based on our previously published study demonstrating that intact structured membrane microdomains (SMDs) are required for NTSR-1 mitogenic signaling, we hypothesized that regulated receptor palmitoylation is responsible for NTSR-1 localization and signaling within SMDs upon NTS stimulation. Site-directed mutagenesis and pharmacological strategies were utilized to assess NTRS-1 post-translational modifications in an overexpression cell model (HE K293T) as well as a native breast cancer cell model (MDA-MB-231). NTSR-1 palmitoylation was confirmed by multiple chemical and fluororadiographic methodologies. NTSR-1 glycosylation was confirmed by pharmacological (tunicamycin) and chemical (PGNaseF and O-type glycosidase) approaches. Physiological correlates including cell viability (MTS assay), apoptosis (caspase-3/7 assay) and ERK phosphorylation were utilized to assess the consequences of NTRS-1 palmitoylation. The interaction between palmitoylated NTRS-1 and Gα q/11 within SMDS was confirmed with immunopreciptation analysis of detergent-free isolated fractions of caveolin-rich microdomains. We identified dual-palmitoylation at Cys381 and Cys383 of endogenously-expressed NTSR-1 in MDA-MB-231 breast adeno-carcinomas as well as exogenously-expressed NTSR-1 in HE K293T cells (which do not normally express NTSR-1). Pharmacological inhibition of NTSR-1 palmitoylation in MDA-MB-231 cells as well as NTSR-1-expressing HE K293T cells diminished NTS-mediated ERK 1/2 phosphorylation. Additionally, NTSR-1 mutated at Cys381 and Cys383 showed diminished ERK1/2 stimulation and reduced ability to protect HE K293T cells against apoptosis induced by serum starvation. Mechanistically, mutated C381,383S-NTSR-1 showed reduced ability to interact with Gα q/11 and diminished localization to structured membrane microdomains (SMDs), where Gα q/11 preferentially resides. We also demonstrated that only glycosylated isoforms of NTRS-1 localize within SMDs by palmitotylation. Collectively, our data establish palmitoylation as a novel pharmacological target to inhibit NTSR-1 mitogenic signaling in breast cancer cells.

Original languageEnglish (US)
Pages (from-to)427-435
Number of pages9
JournalCancer Biology and Therapy
Volume12
Issue number5
DOIs
StatePublished - Sep 1 2011

Fingerprint

Lipoylation
Membrane Microdomains
Breast Neoplasms
Neurotensin
Pharmacology
neurotensin type 1 receptor
Phosphorylation
Apoptosis
Caveolins
Caspase 7
Tunicamycin
Glycoside Hydrolases
Post Translational Protein Processing
G-Protein-Coupled Receptors
Starvation
Site-Directed Mutagenesis
Glycosylation

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Oncology
  • Pharmacology
  • Cancer Research

Cite this

@article{71b9db2aa0ff4b27b5374a4d434a9e11,
title = "Neurotensin receptor-1 inducible palmitoylation is required for efficient receptor-mediated mitogenic-signaling within structured membrane microdomains",
abstract = "Neurotensin receptor-1 (NTSR-1) is a G-protein coupled receptor (GPCR) that has been recently identified as a mediator of cancer progression. NTSR-1 and its endogenous ligand, neurotensin (NTS), are co-expressed in several breast cancer cell lines and breast cancer tumor samples. Based on our previously published study demonstrating that intact structured membrane microdomains (SMDs) are required for NTSR-1 mitogenic signaling, we hypothesized that regulated receptor palmitoylation is responsible for NTSR-1 localization and signaling within SMDs upon NTS stimulation. Site-directed mutagenesis and pharmacological strategies were utilized to assess NTRS-1 post-translational modifications in an overexpression cell model (HE K293T) as well as a native breast cancer cell model (MDA-MB-231). NTSR-1 palmitoylation was confirmed by multiple chemical and fluororadiographic methodologies. NTSR-1 glycosylation was confirmed by pharmacological (tunicamycin) and chemical (PGNaseF and O-type glycosidase) approaches. Physiological correlates including cell viability (MTS assay), apoptosis (caspase-3/7 assay) and ERK phosphorylation were utilized to assess the consequences of NTRS-1 palmitoylation. The interaction between palmitoylated NTRS-1 and Gα q/11 within SMDS was confirmed with immunopreciptation analysis of detergent-free isolated fractions of caveolin-rich microdomains. We identified dual-palmitoylation at Cys381 and Cys383 of endogenously-expressed NTSR-1 in MDA-MB-231 breast adeno-carcinomas as well as exogenously-expressed NTSR-1 in HE K293T cells (which do not normally express NTSR-1). Pharmacological inhibition of NTSR-1 palmitoylation in MDA-MB-231 cells as well as NTSR-1-expressing HE K293T cells diminished NTS-mediated ERK 1/2 phosphorylation. Additionally, NTSR-1 mutated at Cys381 and Cys383 showed diminished ERK1/2 stimulation and reduced ability to protect HE K293T cells against apoptosis induced by serum starvation. Mechanistically, mutated C381,383S-NTSR-1 showed reduced ability to interact with Gα q/11 and diminished localization to structured membrane microdomains (SMDs), where Gα q/11 preferentially resides. We also demonstrated that only glycosylated isoforms of NTRS-1 localize within SMDs by palmitotylation. Collectively, our data establish palmitoylation as a novel pharmacological target to inhibit NTSR-1 mitogenic signaling in breast cancer cells.",
author = "Yasser Heakal and Woll, {Matthew P.} and Todd Fox and Kelly Seaton and Robert Levenson and Mark Kester",
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Neurotensin receptor-1 inducible palmitoylation is required for efficient receptor-mediated mitogenic-signaling within structured membrane microdomains. / Heakal, Yasser; Woll, Matthew P.; Fox, Todd; Seaton, Kelly; Levenson, Robert; Kester, Mark.

In: Cancer Biology and Therapy, Vol. 12, No. 5, 01.09.2011, p. 427-435.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Neurotensin receptor-1 inducible palmitoylation is required for efficient receptor-mediated mitogenic-signaling within structured membrane microdomains

AU - Heakal, Yasser

AU - Woll, Matthew P.

AU - Fox, Todd

AU - Seaton, Kelly

AU - Levenson, Robert

AU - Kester, Mark

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N2 - Neurotensin receptor-1 (NTSR-1) is a G-protein coupled receptor (GPCR) that has been recently identified as a mediator of cancer progression. NTSR-1 and its endogenous ligand, neurotensin (NTS), are co-expressed in several breast cancer cell lines and breast cancer tumor samples. Based on our previously published study demonstrating that intact structured membrane microdomains (SMDs) are required for NTSR-1 mitogenic signaling, we hypothesized that regulated receptor palmitoylation is responsible for NTSR-1 localization and signaling within SMDs upon NTS stimulation. Site-directed mutagenesis and pharmacological strategies were utilized to assess NTRS-1 post-translational modifications in an overexpression cell model (HE K293T) as well as a native breast cancer cell model (MDA-MB-231). NTSR-1 palmitoylation was confirmed by multiple chemical and fluororadiographic methodologies. NTSR-1 glycosylation was confirmed by pharmacological (tunicamycin) and chemical (PGNaseF and O-type glycosidase) approaches. Physiological correlates including cell viability (MTS assay), apoptosis (caspase-3/7 assay) and ERK phosphorylation were utilized to assess the consequences of NTRS-1 palmitoylation. The interaction between palmitoylated NTRS-1 and Gα q/11 within SMDS was confirmed with immunopreciptation analysis of detergent-free isolated fractions of caveolin-rich microdomains. We identified dual-palmitoylation at Cys381 and Cys383 of endogenously-expressed NTSR-1 in MDA-MB-231 breast adeno-carcinomas as well as exogenously-expressed NTSR-1 in HE K293T cells (which do not normally express NTSR-1). Pharmacological inhibition of NTSR-1 palmitoylation in MDA-MB-231 cells as well as NTSR-1-expressing HE K293T cells diminished NTS-mediated ERK 1/2 phosphorylation. Additionally, NTSR-1 mutated at Cys381 and Cys383 showed diminished ERK1/2 stimulation and reduced ability to protect HE K293T cells against apoptosis induced by serum starvation. Mechanistically, mutated C381,383S-NTSR-1 showed reduced ability to interact with Gα q/11 and diminished localization to structured membrane microdomains (SMDs), where Gα q/11 preferentially resides. We also demonstrated that only glycosylated isoforms of NTRS-1 localize within SMDs by palmitotylation. Collectively, our data establish palmitoylation as a novel pharmacological target to inhibit NTSR-1 mitogenic signaling in breast cancer cells.

AB - Neurotensin receptor-1 (NTSR-1) is a G-protein coupled receptor (GPCR) that has been recently identified as a mediator of cancer progression. NTSR-1 and its endogenous ligand, neurotensin (NTS), are co-expressed in several breast cancer cell lines and breast cancer tumor samples. Based on our previously published study demonstrating that intact structured membrane microdomains (SMDs) are required for NTSR-1 mitogenic signaling, we hypothesized that regulated receptor palmitoylation is responsible for NTSR-1 localization and signaling within SMDs upon NTS stimulation. Site-directed mutagenesis and pharmacological strategies were utilized to assess NTRS-1 post-translational modifications in an overexpression cell model (HE K293T) as well as a native breast cancer cell model (MDA-MB-231). NTSR-1 palmitoylation was confirmed by multiple chemical and fluororadiographic methodologies. NTSR-1 glycosylation was confirmed by pharmacological (tunicamycin) and chemical (PGNaseF and O-type glycosidase) approaches. Physiological correlates including cell viability (MTS assay), apoptosis (caspase-3/7 assay) and ERK phosphorylation were utilized to assess the consequences of NTRS-1 palmitoylation. The interaction between palmitoylated NTRS-1 and Gα q/11 within SMDS was confirmed with immunopreciptation analysis of detergent-free isolated fractions of caveolin-rich microdomains. We identified dual-palmitoylation at Cys381 and Cys383 of endogenously-expressed NTSR-1 in MDA-MB-231 breast adeno-carcinomas as well as exogenously-expressed NTSR-1 in HE K293T cells (which do not normally express NTSR-1). Pharmacological inhibition of NTSR-1 palmitoylation in MDA-MB-231 cells as well as NTSR-1-expressing HE K293T cells diminished NTS-mediated ERK 1/2 phosphorylation. Additionally, NTSR-1 mutated at Cys381 and Cys383 showed diminished ERK1/2 stimulation and reduced ability to protect HE K293T cells against apoptosis induced by serum starvation. Mechanistically, mutated C381,383S-NTSR-1 showed reduced ability to interact with Gα q/11 and diminished localization to structured membrane microdomains (SMDs), where Gα q/11 preferentially resides. We also demonstrated that only glycosylated isoforms of NTRS-1 localize within SMDs by palmitotylation. Collectively, our data establish palmitoylation as a novel pharmacological target to inhibit NTSR-1 mitogenic signaling in breast cancer cells.

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