Aim and Objective: Screening of active components from a natural product, especially
from a crude extract, is a great challenge. To avoid potential activity interference of the N-terminus
modification in the most common constructs based on GCPRs labeled with GFP technology, a Cterminus
tGFP-labeled hGLP-1 receptor containing recombinant cell line hGLP-1R-tGFP was
constructed and tried to be used in the screening of natural products from Chinese herb.
Materials and Methods: The GLP1 receptor gene was amplified and the inserts pCMV6-AC-tGFP
and tGFP were fused at the C-terminus of GLP1 receptor to construct a recombinant plasmid. The
recombinant was transfected into U2OS cell and selected with antibiotics and flow cytometry. The
constructed cell line was named as hGLP-1R-tGFP cell line. The expression levels of GLP-1R-tGFP
protein were confirmed by western-blot. The fluorescence imaging of re-distribution from diffusing
to aggregate spots inside the cells was quantitated and analyzed by High Content Screening (HCS)
assay. Meanwhile, the specificity, stability and C-terminus function of hGLP-1R-tGFP cell line were
characterized. In order to allow the recombinant cell line of hGLP-1R-tGFP to be suitable in highcontent
system of Arrayscan-infinity-700 in screening mode, several conditions have also been
optimized. In the end, a total of 100 crude extract samples provided by the Yunnan Institute of
Materia Medica have been screened with this method.
Results: Upon the activation of GLP-1 receptors by Exendin 4, fluorescent patches appeared on the cell
membrane and subsequently internalized to form fluorescent aggregates inside the cells under
fluorescent microscopy examination. The agonistic activity, sensitivity and specificity of the formation
of fluorescent aggregate spot in hGLP-1R-tGFP cells have been confirmed by the activation of GLP-1R
using the GLP-1analogues. The agonistic effects of GLP-1 analogues are blocked by a GLP-1R
antagonist, Exendin9-39. The downstream of GLP-1 pathway, the activation of adenylate cyclase and
the raising of cellular cAMP levels, remained intact in these tGFP modified C-terminus GLP-1 receptor
cells. Meanwhile, a total of 100 crude extract samples from Chinese herbs have been screened by this
method to find new active ingredients.
Conclusion: Combined with High Content Screening image and data automatic acquisition
processing, a new screening assay based on a recombinant U2OS cell line which GFP labeled at the
C terminus of GLP1 receptor has been developed. GLP-1R agonist activity in extracts of Astragalus
propinquus and Panax notoginseng from Chinese herbs has been determined by this method.