Identification of novel transplantable GPCR recycling motif for drug discovery.

β-Adrenergic receptor (β-AR) agonists and antagonists are widely used in the treatment of major cardiovascular diseases such as heart failure and hypertension. The β-AR like other G protein-coupled receptors (GPCRs) are endocytosed in response to intense agonist activation. Recycling of the agonist-internalized β-AR is dependent on its carboxy-terminal type-1 PSD-95/DLG/ZO1 (PDZ) and on phospho-serine in the third intracellular loop of the β-AR. Progressive elongation of the β-AR at its C-tail inactivated the PDZ-biding domain and inhibited the recycling of the β-AR. However, fusing a twenty amino acid peptide derived from the multiple cloning region of the mammalian expression vector pCDNA3 to the C-tail of the β-AR (β-AR[+20]) produced a chimeric β-AR that recycled rapidly and efficiently. The β-AR[+20] recycled in a type-1 PDZ and phospho-Ser-independent manner, indicating that this peptide provided a general GPCR recycling signal. Fusing the enhanced yellow fluorescent protein (EYFP) down-stream of β-AR[+20] generated a β-AR-EYFP chimera that was expressed on the membrane and recycled efficiently after agonist-induced internalization. This construct trafficked in a PDZ-SNX27/retromer-independent manner. We also fused EYFP to the N-terminus of the β-AR to created EYFP-WT β-AR. This construct recycled in PDZ and SNX27/retromer dependent manner. These β-AR-EYFP constructs would be useful for high throughput screening (HTS) programs to identify new entities that would interfere with the recycling of agonist internalized GPCR that traffic in PDZ-dependent vs. PDZ-independent roadmaps.