Cell signaling and trafficking of human melanocortin receptors in real time using two-photon fluorescence and confocal laser microscopy: Differentiation of agonists and antagonists

Minying Cai, Eva V. Varga, Magda Stankova, Alexander Mayorov, Joseph W. Perry, Henry I. Yamamura, Dev Trivedi, Victor J Hruby

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

Melanocortin hormones and neurotransmitters regulate a vast array of physiologic processes by interacting with five G-protein-coupled melanocortin receptor types. In the present study, we have systematically studied the regulation of individual human melanocortin receptor wild subtypes using a synthetic rhodamine-labeled human melanotropin agonist and antagonist, arrestins fused to green fluorescent protein in conjunction with two-photon fluorescence laser scanning microscopy and confocal microscopy. Stimulation of the melanocortin receptors by its cognate agonist triggered rapid arrestin recruitment and receptor internalization for all four human melanocortin receptors examined. Antagonists-bound melanocortin receptors, on the other hand, did not recruit β-arrestins, and remained in the cell membrane even after long-term (30 min) treatment. Agonist-mediated internalization of all melanocortin receptor subtypes was sensitive to inhibitors of clathrin-dependent endocytosis, but not to caveolae inhibitors. In summary, agonist-mediated internalization of all subtypes of melanocortin receptors are dependent upon β-arrestin-mediated clathrin-coated pits, whereas, β-arrestin-2 conjugated green fluorescence protein (β-arrestin-2-GFP) recruitment is not dependent on protein kinase A activation. Real time two-photon fluorescence laser scanning microscopy is a most powerful tool to study the dynamic processes in living cells and tissues, without inflicting significant and often lethal damage to the specimen.

Original languageEnglish (US)
Pages (from-to)183-193
Number of pages11
JournalChemical Biology and Drug Design
Volume68
Issue number4
DOIs
StatePublished - Oct 2006

Fingerprint

Human Trafficking
Melanocortin Receptors
Cell signaling
Photons
Confocal Microscopy
Microscopic examination
Fluorescence
Lasers
Arrestin
Arrestins
Clathrin
Fluorescence Microscopy
Melanocortins
Melanocyte-Stimulating Hormones
Scanning
Caveolae
Rhodamines
Confocal microscopy
Cell membranes
G-Protein-Coupled Receptors

Keywords

  • β-arrestin
  • Green fluorescence protein
  • Melanocortin receptors
  • MTII
  • Rhodamine
  • SHU-9119
  • Two-photon fluorescence laser scanning microscopy

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine

Cite this

Cell signaling and trafficking of human melanocortin receptors in real time using two-photon fluorescence and confocal laser microscopy : Differentiation of agonists and antagonists. / Cai, Minying; Varga, Eva V.; Stankova, Magda; Mayorov, Alexander; Perry, Joseph W.; Yamamura, Henry I.; Trivedi, Dev; Hruby, Victor J.

In: Chemical Biology and Drug Design, Vol. 68, No. 4, 10.2006, p. 183-193.

Research output: Contribution to journalArticle

@article{d10c25df2f2846d5a9a67b6e98395cdb,
title = "Cell signaling and trafficking of human melanocortin receptors in real time using two-photon fluorescence and confocal laser microscopy: Differentiation of agonists and antagonists",
abstract = "Melanocortin hormones and neurotransmitters regulate a vast array of physiologic processes by interacting with five G-protein-coupled melanocortin receptor types. In the present study, we have systematically studied the regulation of individual human melanocortin receptor wild subtypes using a synthetic rhodamine-labeled human melanotropin agonist and antagonist, arrestins fused to green fluorescent protein in conjunction with two-photon fluorescence laser scanning microscopy and confocal microscopy. Stimulation of the melanocortin receptors by its cognate agonist triggered rapid arrestin recruitment and receptor internalization for all four human melanocortin receptors examined. Antagonists-bound melanocortin receptors, on the other hand, did not recruit β-arrestins, and remained in the cell membrane even after long-term (30 min) treatment. Agonist-mediated internalization of all melanocortin receptor subtypes was sensitive to inhibitors of clathrin-dependent endocytosis, but not to caveolae inhibitors. In summary, agonist-mediated internalization of all subtypes of melanocortin receptors are dependent upon β-arrestin-mediated clathrin-coated pits, whereas, β-arrestin-2 conjugated green fluorescence protein (β-arrestin-2-GFP) recruitment is not dependent on protein kinase A activation. Real time two-photon fluorescence laser scanning microscopy is a most powerful tool to study the dynamic processes in living cells and tissues, without inflicting significant and often lethal damage to the specimen.",
keywords = "β-arrestin, Green fluorescence protein, Melanocortin receptors, MTII, Rhodamine, SHU-9119, Two-photon fluorescence laser scanning microscopy",
author = "Minying Cai and Varga, {Eva V.} and Magda Stankova and Alexander Mayorov and Perry, {Joseph W.} and Yamamura, {Henry I.} and Dev Trivedi and Hruby, {Victor J}",
year = "2006",
month = "10",
doi = "10.1111/j.1747-0285.2006.00432.x",
language = "English (US)",
volume = "68",
pages = "183--193",
journal = "Chemical Biology and Drug Design",
issn = "1747-0277",
publisher = "Blackwell",
number = "4",

}

TY - JOUR

T1 - Cell signaling and trafficking of human melanocortin receptors in real time using two-photon fluorescence and confocal laser microscopy

T2 - Differentiation of agonists and antagonists

AU - Cai, Minying

AU - Varga, Eva V.

AU - Stankova, Magda

AU - Mayorov, Alexander

AU - Perry, Joseph W.

AU - Yamamura, Henry I.

AU - Trivedi, Dev

AU - Hruby, Victor J

PY - 2006/10

Y1 - 2006/10

N2 - Melanocortin hormones and neurotransmitters regulate a vast array of physiologic processes by interacting with five G-protein-coupled melanocortin receptor types. In the present study, we have systematically studied the regulation of individual human melanocortin receptor wild subtypes using a synthetic rhodamine-labeled human melanotropin agonist and antagonist, arrestins fused to green fluorescent protein in conjunction with two-photon fluorescence laser scanning microscopy and confocal microscopy. Stimulation of the melanocortin receptors by its cognate agonist triggered rapid arrestin recruitment and receptor internalization for all four human melanocortin receptors examined. Antagonists-bound melanocortin receptors, on the other hand, did not recruit β-arrestins, and remained in the cell membrane even after long-term (30 min) treatment. Agonist-mediated internalization of all melanocortin receptor subtypes was sensitive to inhibitors of clathrin-dependent endocytosis, but not to caveolae inhibitors. In summary, agonist-mediated internalization of all subtypes of melanocortin receptors are dependent upon β-arrestin-mediated clathrin-coated pits, whereas, β-arrestin-2 conjugated green fluorescence protein (β-arrestin-2-GFP) recruitment is not dependent on protein kinase A activation. Real time two-photon fluorescence laser scanning microscopy is a most powerful tool to study the dynamic processes in living cells and tissues, without inflicting significant and often lethal damage to the specimen.

AB - Melanocortin hormones and neurotransmitters regulate a vast array of physiologic processes by interacting with five G-protein-coupled melanocortin receptor types. In the present study, we have systematically studied the regulation of individual human melanocortin receptor wild subtypes using a synthetic rhodamine-labeled human melanotropin agonist and antagonist, arrestins fused to green fluorescent protein in conjunction with two-photon fluorescence laser scanning microscopy and confocal microscopy. Stimulation of the melanocortin receptors by its cognate agonist triggered rapid arrestin recruitment and receptor internalization for all four human melanocortin receptors examined. Antagonists-bound melanocortin receptors, on the other hand, did not recruit β-arrestins, and remained in the cell membrane even after long-term (30 min) treatment. Agonist-mediated internalization of all melanocortin receptor subtypes was sensitive to inhibitors of clathrin-dependent endocytosis, but not to caveolae inhibitors. In summary, agonist-mediated internalization of all subtypes of melanocortin receptors are dependent upon β-arrestin-mediated clathrin-coated pits, whereas, β-arrestin-2 conjugated green fluorescence protein (β-arrestin-2-GFP) recruitment is not dependent on protein kinase A activation. Real time two-photon fluorescence laser scanning microscopy is a most powerful tool to study the dynamic processes in living cells and tissues, without inflicting significant and often lethal damage to the specimen.

KW - β-arrestin

KW - Green fluorescence protein

KW - Melanocortin receptors

KW - MTII

KW - Rhodamine

KW - SHU-9119

KW - Two-photon fluorescence laser scanning microscopy

UR - http://www.scopus.com/inward/record.url?scp=33751048740&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33751048740&partnerID=8YFLogxK

U2 - 10.1111/j.1747-0285.2006.00432.x

DO - 10.1111/j.1747-0285.2006.00432.x

M3 - Article

C2 - 17105482

AN - SCOPUS:33751048740

VL - 68

SP - 183

EP - 193

JO - Chemical Biology and Drug Design

JF - Chemical Biology and Drug Design

SN - 1747-0277

IS - 4

ER -