title: Identification of novel transplantable GPCR recycling motif for drug discovery.
abstract: β1-Adrenergic receptor (β1-AR) agonists and antagonists are widely used in the treatment of major cardiovascular diseases such as heart failure and hypertension. The β1-AR like other G protein-coupled receptors (GPCRs) are endocytosed in response to intense agonist activation. Recycling of the agonist-internalized β1-AR is dependent on its carboxy-terminal type-1 PSD-95/DLG/ZO1 (PDZ) and on phospho-serine312in the third intracellular loop of the β1-AR. Progressive elongation of the β1-AR at its C-tail inactivated the PDZ-biding domain and inhibited the recycling of the β1-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 β1-AR (β1-AR[+20]) produced a chimeric β1-AR that recycled rapidly and efficiently. The β1-AR[+20] recycled in a type-1 PDZ and phospho-Ser312-independent manner, indicating that this peptide provided a general GPCR recycling signal. Fusing the enhanced yellow fluorescent protein (EYFP) down-stream of β1-AR[+20] generated a β1-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 β1-AR to created EYFP-WT β1-AR. This construct recycled in PDZ and SNX27/retromer dependent manner. These β1-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.
authors.affiliation: Department of Pharmacology, The University of Tennessee Health Sciences Center, 71 S. Manassas, Memphis, TN 38103, USA; Department of Biochemistry, Faculty of Pharmacy Cairo University, Kasr El-Aini St., Cairo 11562, Egypt.
authors.forename: Mohammed M
authors.initials: MM
authors.lastname: Nooh
authors.affiliation: Department of Physiology, The University of Tennessee Health Sciences Center, 71 S. Manassas, Memphis, TN 38103, USA.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Mancarella
authors.affiliation: Department of Pharmacology, The University of Tennessee Health Sciences Center, 71 S. Manassas, Memphis, TN 38103, USA. Electronic address: sbahouth@uthsc.edu.
authors.forename: Suleiman W
authors.initials: SW
authors.lastname: Bahouth
mesh_terms: D058665:Adrenergic beta-1 Receptor Agonists / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D055808:Drug Discovery / Q000379:methods*; D004705:Endocytosis / Q000187:drug effects*; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D054731:PDZ Domains; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010768:Phosphoserine / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D018342:Receptors, Adrenergic, beta-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D058305:Sorting Nexins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C547787:ADRB1 protein, human; D058665:Adrenergic beta-1 Receptor Agonists; D001426:Bacterial Proteins; D008164:Luminescent Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D018342:Receptors, Adrenergic, beta-1; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; C522885:SNX27 protein, human; D058305:Sorting Nexins; C054562:yellow fluorescent protein, Bacteria; D010768:Phosphoserine; C085706:FLAG peptide
keywords: Confocal microscopy; GPCR; PDZ; SNX27; Trafficking; β-Adrenergic receptors