Identification of novel transplantable GPCR recycling motif for drug discovery

Research output: Contribution to journalArticle

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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-serine312 in 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.

Original languageEnglish (US)
Pages (from-to)22-32
Number of pages11
JournalBiochemical Pharmacology
Volume120
DOIs
StatePublished - Nov 15 2016

Fingerprint

Recycling
Drug Discovery
G-Protein-Coupled Receptors
Adrenergic Receptors
Proteins
Adrenergic Agonists
Peptides
Adrenergic Antagonists
Cloning
Endocytosis
Organism Cloning
Elongation
Screening
Cardiovascular Diseases
Heart Failure

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Pharmacology

Cite this

Identification of novel transplantable GPCR recycling motif for drug discovery. / Nooh, Mohammed M.; Mancarella, Salvatore; Bahouth, Suleiman.

In: Biochemical Pharmacology, Vol. 120, 15.11.2016, p. 22-32.

Research output: Contribution to journalArticle

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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-serine312 in 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.",
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