| 摘要 |
There is disclosed a method of determining whether a candidate compound mimics or antagonizes effects of insulin, comprising: (a) providing a transfected host cell comprising a nucleic acid sequence which encodes a protein construct comprising an IRAP protein moiety and a detectable protein moiety, wherein the detectable protein moiety is fused to a luminal domain of the IRAP protein moiety and further wherein the detectable protein moiety is capable of being assayed by a first detection method capable of detecting the protein moiety when located exofacially but not when located intracellularly and a second detection method capable of detecting the protein moiety both when located exofacially and when located intracellularly; (b) contacting the transfected host cell with a candidate compound; (c) measuring the amount of the exofacial protein moiety by the said first detection method and measuring the total amount of exofacial and intracellular detectable protein moiety by the said second detection method; and (d) comparing the amount of exofacial detectable protein moiety with the total amount of exofacial and intracellular detectable protein moiety to provide a measure of the extent of GLUT4 vesicle translocation. In an alternative method, the protein construct comprises an IRAP protein moiety, a first detectable protein moiety which is fused to a luminal domain of the IRAP protein moiety and a second detectable protein moiety which is fused to a cytoplasmic domain of the IRAP protein moiety, wherein the first detectable protein moiety is capable of being assayed by a first detection method capable of detecting the first protein moiety when located exofacially but not when located intracellularly and wherein the second detectable protein moiety is capable of being assayed by a second detection method capable of detecting the second protein moiety when located intracellularly. The assays of the invention may be used to determine whether a "candidate compound" is a mimic or antagonist of insulin. The present invention may be used to test libraries of compounds in an automated high throughput screen.
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