Functional expression of recombinant G-protein-coupled receptors monitored by video imaging of pigment movement in melanophores.
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 beta 2-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.
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06536-0812.
T S
TS
McClintock
G F
GF
Graminski
M N
MN
Potenza
C K
CK
Jayawickreme
A
A
Roby-Shemkovitz
M R
MR
Lerner
D000818:Animals; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000235:genetics / Q000502:physiology; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008547:Melanophores / Q000737:chemistry* / Q000502:physiology; D010860:Pigments, Biological / Q000737:chemistry* / Q000502:physiology; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis; D014741:Video Recording; D014982:Xenopus laevis
D010860:Pigments, Biological; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins