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 language | English (US) |
|---|---|
| Pages (from-to) | 427-435 |
| Number of pages | 9 |
| Journal | Cancer Biology and Therapy |
| Volume | 12 |
| Issue number | 5 |
| DOIs | |
| State | Published - Sep 1 2011 |
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All Science Journal Classification (ASJC) codes
- Molecular Medicine
- Oncology
- Pharmacology
- Cancer Research
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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 journal › Article
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
PY - 2011/9/1
Y1 - 2011/9/1
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.
UR - http://www.scopus.com/inward/record.url?scp=80052372651&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=80052372651&partnerID=8YFLogxK
U2 - 10.4161/cbt.12.5.15984
DO - 10.4161/cbt.12.5.15984
M3 - Article
C2 - 21725197
AN - SCOPUS:80052372651
VL - 12
SP - 427
EP - 435
JO - Cancer Biology and Therapy
JF - Cancer Biology and Therapy
SN - 1538-4047
IS - 5
ER -