Multi-transmembrane proteins are far more challenging to express in mammalian cells than cytosolic or single-pass transmembrane proteins. G protein-coupled receptors (GPCRs) are seven-transmembrane spanners. A key question to ask when engineering mammalian cells to express GPCRs is “What’s the right expression level?” The answer is always “It depends!”
More specifically, for immunizing animals to generate GPCR antibodies or screening GPCR antibodies, small molecules or peptide binders, high cell surface expression is generally beneficial. In contrast, it is far more complex when it comes to developing stable cell lines for the purpose of cell-based assays. Depending on the modality of the high throughput screen and the pharmacology of each GPCR, different expression levels may be appropriate for different GPCRs and different screens. There isn’t a one size fits all.
To meet the needs of various drug discovery and research interests, Multispan developed its own patented proprietary vectors, designed for robust surface expression of GPCRs and other multi-transmembrane proteins in mammalian cells. Combined with Multispan’s in-depth know-how in cell engineering and single cell cloning, these proprietary vectors are part of an innovative, powerful and versatile GPCR expression technology platform.
Example 1. We have been tasked with generating stable cell line clones that can clearly discern differences between full and partial agonists for a variety of GPCR targets, while maintaining confident assay windows for partial agonists. One of such example was to engineer a stable cell line for assaying Dopamine receptor D1. To do this, we cloned the full-length cDNA of Dopamine receptor D1 receptor into our proprietary expression vector, transfected and cloned stable cell lines in 96-well microplates, and carefully selected functional clones while monitoring surface expression of the D1 receptor.
Since the Dopamine D1 receptor is coupled via Gαs, the functional assays were performed using full agonists (Dopamine and Fenoldopam), and partial agonist (Bromocriptine) to induce cAMP level increase in the cells using MULTISCREENTM TR-FRET cAMP assay. The same clones were also evaluated by surface expression using flow cytometry. 4 clones representing a gradient of receptor surface expression were obtained are illustrated here. In the lowest D1 receptor expression clone, Clone #4, Bromocriptine failed to induce dose-dependent cAMP activity whereas Dopamine and Fenoldopam induced cAMP with full efficacy. In Clone #32 that has a slightly higher D1 receptor surface expression, Dopamine and Fenoldopam induced full cAMP increase while Bromocriptine induced partial but clearly discernible cAMP increase with lower potency and efficacy compared to the two full agonists. However, in the 2 clones with even higher expression of D1 receptor, Clones #19 and #21, the efficacy separation between Bromocriptine and Dopamine/Fenoldopam disappeared.
As demonstrated by this example, Multispan’s GPCR expression technology platform has the versatility and power to address a variety of cell-based assay needs, where the intricacies of receptor expression levels are key factors in determining success.
Example 2. Generating antibodies that can modulate GPCR function hasn’t been an easy quest. The challenge comes from limited accessibility of functional epitopes, the requirement of membrane for the integrity of the 7-transmembrane GPCR protein structure, and the relative high homology of GPCRs to animal orthologs throughout evolution. Cell-based immunization with overexpressed GPCRs has been shown to effectively induce immune response. To use cells as immunogen, high expression of the membrane target is critical. The Multispan team has been able to leverage our proprietary expression platform to generate high expression cell lines that produce over 1 million GPCR molecules per cell surface, when such extreme high expression is beneficial. These high expression cell lines can not only serve as effective immunogens but also as dynamic tools in binding assays, including radioligand membrane binding, cell-based ELISA and FACS single cell binding.
As examples, we cloned EDG4 (LPA2) and NMUR1 cDNAs into our proprietary expression vector, transfected CHO-K1 and HEK-293T cells and selected single cell clones by high cell surface expression using flow cytometry. The high expression cell line clones have also been validated in the functional assay using ligand-induced Calcium immobilization using MULTISCREENTM Calcium Assay. The results confirmed functional integrity of the GPCRs expressed at high level.
Multispan’s GPCR expression technology platform can robustly create stable cell lines expressing high levels of functional receptors to meet the needs of antibody generation and high throughput binding assays.