title: Endogenous N-terminal Domain Cleavage Modulates α1D-Adrenergic Receptor Pharmacodynamics.
abstract: The α1D-adrenergic receptor (ADRA1D) is a key regulator of cardiovascular, prostate, and central nervous system functions. This clinically relevant G protein-coupled receptor has proven difficult to study, as it must form an obligate modular homodimer containing the PDZ proteins scribble and syntrophin or become retained in the endoplasmic reticulum as non-functional protein. We previously determined that targeted removal of the N-terminal (NT) 79 amino acids facilitates ADRA1D plasma membrane expression and agonist-stimulated functional responses. However, whether such an event occurs in physiological contexts was unknown. Herein, we report the ADRA1D is subjected to innate NT processing in cultured human cells. SNAP near-infrared imaging and tandem-affinity purification revealed the ADRA1D is expressed as both full-length and NT truncated forms in multiple human cell lines. Serial truncation mapping identified the cleavage site as Leu(90)/Val(91) in the 95-amino acid ADRA1D NT domain, suggesting human cells express a Δ1-91 ADRA1D species. Tandem-affinity purification MS/MS and co-immunoprecipitation analysis indicate NT processing of ADRA1D is not required to form scribble-syntrophin macromolecular complexes. Yet, label-free dynamic mass redistribution signaling assays demonstrate that Δ1-91 ADRA1D agonist responses were greater than WT ADRA1D. Mutagenesis of the cleavage site nullified the processing event, resulting in ADRA1D agonist responses less than the WT receptor. Thus, we propose that processing of the ADRA1D NT domain is a physiological mechanism employed by cells to generate a functional ADRA1D isoform with optimal pharmacodynamic properties.
authors.affiliation: the Departments of Pharmacology and.
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Kountz
authors.affiliation: the Departments of Pharmacology and.
authors.forename: Kyung-Soon
authors.initials: KS
authors.lastname: Lee
authors.affiliation: the Departments of Pharmacology and From the Howard Hughes Medical Institute and.
authors.forename: Stacey
authors.initials: S
authors.lastname: Aggarwal-Howarth
authors.affiliation: the Departments of Pharmacology and.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Curran
authors.affiliation: the Departments of Pharmacology and.
authors.forename: Ji-Min
authors.initials: JM
authors.lastname: Park
authors.affiliation: the Departments of Pharmacology and.
authors.forename: Dorathy-Ann
authors.initials: DA
authors.lastname: Harris
authors.affiliation: the Departments of Pharmacology and.
authors.forename: Aaron
authors.initials: A
authors.lastname: Stewart
authors.affiliation: the Departments of Pharmacology and.
authors.forename: Joseph
authors.initials: J
authors.lastname: Hendrickson
authors.affiliation: Genome Sciences, University of Washington School of Medicine, Seattle, Washington 98195.
authors.forename: Nathan D
authors.initials: ND
authors.lastname: Camp
authors.affiliation: Genome Sciences, University of Washington School of Medicine, Seattle, Washington 98195.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Wolf-Yadlin
authors.affiliation: the Departments of Pharmacology and.
authors.forename: Edith H
authors.initials: EH
authors.lastname: Wang
authors.affiliation: the Departments of Pharmacology and From the Howard Hughes Medical Institute and.
authors.forename: John D
authors.initials: JD
authors.lastname: Scott
authors.affiliation: the Departments of Pharmacology and chague@uw.edu.
authors.forename: Chris
authors.initials: C
authors.lastname: Hague
mesh_terms: D056945:Hep G2 Cells; D006801:Humans; D061986:MCF-7 Cells; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D054731:PDZ Domains; D059748:Proteolysis; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics / Q000378:metabolism*
chemical_list: C547719:ADRA1D protein, human; D009363:Neoplasm Proteins; D018340:Receptors, Adrenergic, alpha-1
keywords: G protein-coupled receptor (GPCR); N-terminus; PDZ domain; adrenergic receptor; alpha1D adre; catecholamine; cell signaling; cell surface receptor; label-free signaling; molecular pharmacology