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 The examples described herein are β2-adrenergic receptors whose G-protein coupling domains have been replaced by homologous domains of olfactory receptors, but the procedure has also been used to produce constructs with mutations, deletions, and fusions of two complete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.
|Number of pages||10|
|Journal||Brain Research Protocols|
|State||Published - Dec 1 1997|
Bibliographical noteFunding Information:
We thank T. Landers for critical review of the manuscript and for technical assistance. Supported by Research Grant DC 02736 from the National Institute on Deafness and Other Communication Disorders, National Institutes of Health to T.S.McC. and an Office of Naval Research Grant to M.R.L.
- G-protein coupled receptor
- Signal transduction
ASJC Scopus subject areas
- Neuroscience (all)