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GPCR Stable Cell Line Development

Introduction

G protein–coupled receptors (GPCRs) are a large family of seven transmembrane protein receptors found in eukaryotes. Coupling with G proteins, these receptors are activated by binding to diverse extracellular ligands including light-sensitive compounds, odors, pheromones, hormones, and neurotransmitters etc. The activated GPCRs further induce intracellular signal transduction pathways and, ultimately, cellular responses. GPCRs are involved in a wide variety of physiological processes and many diseases. To date, many medicinal drugs are GPCR-targeting, and there is an increasing interest in the study of discovering novel GPCR-related drug candidates.

Leading Capability

Engineered cell lines stably overexpressing GPCRs are useful models for detection of GPCR signaling. Creative Biogene owns a team of experienced GPCR biologists working in the area of GPCR biology and drug discovery. We have generated a wide variety of GPCR stable cells with more under development. Our expert team and facilities enable us to efficiently construct a GPCR stable cell line to meet your specific needs in the process of GPCR exploration.

With years of experience and thousands of accomplished projects, Creative Biogene offers flexible GPCR detection solutions which can help customer to achieve an ideal GPCR stable cell line suitable for downstream cell-based assay. We can generate stable cell lines used to measure GPCR signaling through second messenger, arrestin recruitment, and internalization readouts.

GPCR Stable Cell Line Development

1) Second Messenger-Mediated Pathway

GPCR activation can be monitored by second messengers such as cAMP and calcium. Upon treatment of ligands, the intracellular cAMP or calcium is changed in a GPCR stable cell line which can be detected using cAMP or calcium cell assay kits. To further simplify and visualize the change of second messengers induced by GPCR activities, Creative Biogene has developed a type of stable cell line containing both a GPCR protein and a fluorescent-based reporter system. Ligand induced GPCR modulation can be measured through fluorescence intensity readout.

2) β-arrestin Recruitment Pathway

β-arrestin is a key protein involved in GPCR signal transduction as a negative regulator. GPCR activation stimulates the β-arrestin to translocate and interact with the activated receptor. This interaction leads to the attenuation of GPCR signaling. The ligand-induced β-arrestin activities have been utilized by our scientists for GPCR stable cell line development. In these cell lines, ligand binding triggers β-arrestin recruitment which can be measured through fluorescent or chemiluminescent output.

3) GPCR Internalization

GPCR internalization is a process of endocytosis induced by ligand binding. Creative Biogene can generate stable cell lines for monitoring ligand-induced GPCR internalization. By fusing a fluorescent protein to the GPCR or based on enzyme fragment complementation, the receptor internalization can be detected by fluorescent or chemiluminescent imaging.

Applications

  • Investigation of GPCR signaling pathways
  • Screening of GPCR agonists and antagonists
  • High throughput screening and compound profiling

Highlights

  • Covering a large broad of GPCR targets from human, mouse, rats and other species
  • Flexibility in cell types such as HEK293, CHO-K1, U-2 OS, 1321N1 etc.
  • Stability through 10 passages
  • Multiplex assays in single cell line
  • Easy detection via commercial assay kits, fluorescent or chemiluminescent imaging
  • Competitive price and quick turnaround time

References:

  1. Rubenstein LA, Lanzara RG (1998). "Activation of G protein-coupled receptors entails cysteine modulation of agonist binding". Journal of Molecular Structure (Theochem). 430: 57–71.
  2. Luttrell LM, Lefkowitz RJ (February 2002). "The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals". Journal of Cell Science. 115 (Pt 3): 455–65.
  3. King N, Hittinger CT, Carroll SB (July 2003). "Evolution of key cell signaling and adhesion protein families predates animal origins". Science. 301 (5631): 361–3.
  4. Penela P, Ribas C, Mayor F (November 2003). "Mechanisms of regulation of the expression and function of G protein-coupled receptor kinases". Cellular Signalling. 15 (11): 973–81.
  5. Filmore D (2004). "It's a GPCR world". Modern Drug Discovery. American Chemical Society. 2004 (November): 24–28.
  6. Tan CM, Brady AE, Nickols HH, Wang Q, Limbird LE (2004). "Membrane trafficking of G protein-coupled receptors". Annual Review of Pharmacology and Toxicology. 44 (1): 559–609.
  7. Overington JP, Al-Lazikani B, Hopkins AL (December 2006). "How many drug targets are there?". Nature Reviews. Drug Discovery. 5 (12): 993–6.
  8. Dorsam RT, Gutkind JS (February 2007). "G-protein-coupled receptors and cancer". Nature Reviews. Cancer. 7 (2): 79–94.

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