TY - JOUR
T1 - Functional expression of recombinant G-protein-coupled receptors monitored by video imaging of pigment movement in melanophores
AU - McClintock, Timothy S.
AU - Graminski, Gerard F.
AU - Potenza, Marc N.
AU - Jayawickreme, Channa K.
AU - Roby-Shemkovitz, Alison
AU - Lerner, Michael R.
PY - 1993
Y1 - 1993
N2 - Xenopus laevis melanophores are capable of functionally expressing recombinant receptors which couple via G-proteins to adenylate cyclase or phospholipase C (PLC). Receptor-mediated stimulation of either of these enzymes causes dispersion of melanosomes while receptor stimulation leading to inhibition of adenylate cyclase induces their aggregation. Translocation of melanosomes within thousands of individual pigment cells was simultaneously tracked by capturing gray scale video images before and after receptor activation. Digital subtraction of poststimulation from prestimulation images was performed on a microcomputer, generating bitplane images containing pixels with nonzero gray scale values wherever melanosome movement had occurred. Movement in both centripetal and centrifugal directions was detectable. The assay was tested using four receptors: A human β2-adrenergic receptor which stimulates adenylate cyclase, murine substance P and bombesin receptors which stimulate PLC, and a human D2 dopamine receptor which inhibits adenylate cyclase. Based on melanosome translocation following application of ligands, expression of functional receptors could be consistently detected in melanophores which received only single copies of a plasmid encoding any of the four receptors. By imaging fields containing up to 11,000 melanophores, the presence of a plasmid coding for a receptor could be detected when its frequency was one per 10,000 plasmids transfected. Combining receptor-mediated pigment translocation in melanophores with the rapid data-handling ability of video technology should provide a bioassay useful for investigating the function of G-protein-coupled receptors and for screening cDNA libraries for clones encoding new receptors.
AB - Xenopus laevis melanophores are capable of functionally expressing recombinant receptors which couple via G-proteins to adenylate cyclase or phospholipase C (PLC). Receptor-mediated stimulation of either of these enzymes causes dispersion of melanosomes while receptor stimulation leading to inhibition of adenylate cyclase induces their aggregation. Translocation of melanosomes within thousands of individual pigment cells was simultaneously tracked by capturing gray scale video images before and after receptor activation. Digital subtraction of poststimulation from prestimulation images was performed on a microcomputer, generating bitplane images containing pixels with nonzero gray scale values wherever melanosome movement had occurred. Movement in both centripetal and centrifugal directions was detectable. The assay was tested using four receptors: A human β2-adrenergic receptor which stimulates adenylate cyclase, murine substance P and bombesin receptors which stimulate PLC, and a human D2 dopamine receptor which inhibits adenylate cyclase. Based on melanosome translocation following application of ligands, expression of functional receptors could be consistently detected in melanophores which received only single copies of a plasmid encoding any of the four receptors. By imaging fields containing up to 11,000 melanophores, the presence of a plasmid coding for a receptor could be detected when its frequency was one per 10,000 plasmids transfected. Combining receptor-mediated pigment translocation in melanophores with the rapid data-handling ability of video technology should provide a bioassay useful for investigating the function of G-protein-coupled receptors and for screening cDNA libraries for clones encoding new receptors.
UR - http://www.scopus.com/inward/record.url?scp=0027156062&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0027156062&partnerID=8YFLogxK
U2 - 10.1006/abio.1993.1123
DO - 10.1006/abio.1993.1123
M3 - Article
C2 - 8385890
AN - SCOPUS:0027156062
SN - 0003-2697
VL - 209
SP - 298
EP - 305
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -