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A novel expression system for Gs-coupled receptors

L Catanzariti¹, J M Allen, B A Hemmings

¹Friedrich Miescher-Institut, Basel, Switzerland.

|September 1, 1993

Summary

LLC-PK1 cells can detect new receptors. Researchers used these cells to identify beta 2 adrenergic receptors (beta 2AR) by measuring urokinase-type plasminogen activator (uPA) secretion, proving the system

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Area of Science:

Cell Biology
Molecular Pharmacology
Biochemistry

Background:

Renal epithelial LLC-PK1 cells exhibit high urokinase-type plasminogen activator (uPA) secretion upon stimulation via the cAMP signaling pathway.
This characteristic response can be leveraged for identifying cells expressing specific receptors.
Gs-protein coupled receptors (Gs-GPCRs) are a significant class of cell surface receptors involved in numerous physiological processes.

Purpose of the Study:

To establish a novel screening system for identifying LLC-PK1-derived cell lines expressing functional heterologous Gs-protein coupled receptors.
To demonstrate the utility of this system by successfully identifying and characterizing cells expressing the mouse beta 2 adrenergic receptor (beta 2AR).

Main Methods:

Stable transfection of LLC-PK1 cells with a mouse beta 2AR genomic clone.
Stimulation of transfected cells with isoproterenol, a beta 2AR agonist.
Screening for increased uPA secretion as an indicator of functional beta 2AR expression.
Confirmation of beta 2AR expression and function through radioligand binding assays and mRNA analysis.

Main Results:

LLC-PK1 cells successfully expressing functional mouse beta 2AR were identified.
These positive clones exhibited dose-dependent uPA secretion in response to isoproterenol.
Specific binding of the beta 2AR-agonist iodocyanopindolol confirmed receptor presence.
Expression of beta 2AR mRNA validated successful gene transfection and expression.

Conclusions:

The developed system effectively utilizes inducible uPA secretion in LLC-PK1 cells to assay for beta 2AR and beta 2AR-agonist function.
This platform is suitable for the functional expression and screening of other heterologous receptors that couple to Gs-proteins.
This methodology offers a robust approach for receptor discovery and characterization in cell-based assays.

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