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Cell Signaling and Trafficking of Human Melanocortin Receptors in Real Time Using Two‐photon Fluorescence and Confocal Laser Microscopy: Differentiation of Agonists and Antagonists
Author(s) -
Cai Minying,
Varga Eva V.,
Stankova Magda,
Mayorov Alexander,
Perry Joseph W.,
Yamamura Henry I.,
Trivedi Dev,
Hruby Victor J.
Publication year - 2006
Publication title -
chemical biology and drug design
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.59
H-Index - 77
eISSN - 1747-0285
pISSN - 1747-0277
DOI - 10.1111/j.1747-0285.2006.00432.x
Subject(s) - melanocortin , internalization , receptor , microbiology and biotechnology , arrestin , agonist , g protein coupled receptor , melanocortin 3 receptor , biology , biophysics , chemistry , melanocortin receptor , signal transduction , biochemistry
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.