Functional analysis by imaging of melanophore pigment dispersion of chimeric receptors constructed by recombinant polymerase chain reaction.
Analysis of functional aspects of the molecular structure of proteins often requires a means to selectively alter structure and subsequently analyze function. We have adapted a method of overlap extension polymerase chain reaction (PCR) to generate multiple domain replacements in G-protein coupled receptors. The examples described herein are beta 2-adrenergic receptors whose G-protein coupling domains have been replaced by homologous domains of olfactory receptors, but the procedure has also been used to produce constructs with mutations, deletions, and fusions of two complete open reading frames. The chimeric olfactory-adrenergic receptors were assayed by functional expression in clonal lines of Xenopus melanophores. The ability of G-protein coupled second messenger pathways to cause translocation of pigment organelles within melanophores allows the use of video microscopy to assay the function of the chimeric receptors. Digital automation of microscope stage, camera, and image processing allows multiple parallel experiments to be performed. Melanophores allow responses mediated by the Gs, Gq and Gi pathways to be assayed with equal efficiency and the specificity of the coupling between chimera (or receptor) and G-protein subtypes can be rapidly determined.
McClintock
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Department of Physiology, University of Kentucky, Lexington 40536-0084, USA. mcclint@pop.uky.edu
Lerner
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D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002999:Clone Cells; D017931:DNA Primers; D004274:DNA, Recombinant; D019204:GTP-Binding Proteins / Q000378:metabolism; D007202:Indicators and Reagents; D008547:Melanophores / Q000166:cytology* / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D018343:Receptors, Adrenergic, beta-2 / Q000032:analysis / Q000096:biosynthesis*; D018035:Receptors, Odorant / Q000032:analysis / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D014162:Transfection / Q000379:methods; D014981:Xenopus
D017931:DNA Primers; D004274:DNA, Recombinant; D007202:Indicators and Reagents; D018343:Receptors, Adrenergic, beta-2; D018035:Receptors, Odorant; D011993:Recombinant Fusion Proteins; D019204:GTP-Binding Proteins