Endogenous N-terminal Domain Cleavage Modulates α1D-Adrenergic Receptor Pharmacodynamics

Abstract

The ␣ 1D -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 nonfunctional 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 fulllength 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 ⌬1-91 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 ⌬1-91 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.␣ 1 -Adrenergic receptors (ARs) 6 belong to the superfamily of class A G protein-coupled receptors (GPCRs). Stimulated by the endogenous catecholamines norepinephrine and epinephrine, ␣ 1 -ARs help coordinate sympathetic nervous function along with the ␣ 2 -and ␤-AR subtypes. This mode of GPCR signaling is particularly relevant during stress, exercise, or lifethreatening situations, as it permits an organism to respond to environmental stimuli supra-maximally and thereby enhance survival probability.Significant gaps remain in our understanding of ␣ 1 -AR biology. Of particular note is the ␣ 1D -AR subtype (ADRA1D). Unlike the closely related ␣ 1A (ADRA1A) and ␣ 1B (ADRA1B) subtypes, which achieve significant plasma membrane expression and robustly respond to agonists in cultured cells, the ADRA1D is cumbersome to study in vitro (1). Following its initial cloning and pharmacological characterization (2-4), numerous studies revealed the ADRA1D is sequestered intracellularly in myriad cell lines (5-11), where it has limited access to agonists and displays minimal functional activity. In a key study, Fan et al. (12) demonstrated ADRA1D functional expression is lost in cultured aortic vascular muscle cells ϳ48 h postdissection. They concluded that factors required for ADRA1D functional expression in vivo are absent in cell culture conditions (12), which may explain ...

The E2.65A mutation disrupts dynamic binding poses of SB269652 at the dopamine D2 and D3 receptors

“…In all conditions, the NT forms a lid over the extracellular vestibule and is in direct interaction with SB269652, suggesting a previously unappreciated role of the NT in ligand binding at D2R and D3R. Indeed, the functional roles of the NT have been documented recently in a few closely related homologs, including the α 1D -adrenergic [ 27 ], 5-HT 2B [ 28 ] and μ-opioid receptors [ 29 , 30 ]. By systematically examining all the available high-resolution crystal structures of class-A GPCRs bound to small compounds, we found 9 structures of 6 receptors showing direct interactions (within 5 Å of the heavy atoms) between NT residues and the small-molecule ligands that at least partially occupy the OBS.…”

Updates in the function and regulation of α1‐adrenoceptors

“…In recombinant systems, α 1D ‐ adrenoceptors are mainly misfolded or unable to undergo proper ligand binding and intracellularly located due to a motif present within the first N‐terminal 79 amino acids of the receptor (Hague, Chen, et al, ; Hague, Uberti, et al, ; Pupo, Uberti, & Minneman, ). Recent evidence has indicated that N‐terminal proteolysis of the α 1D ‐ adrenoceptors could be an endogenous mechanism for proper plasma membrane expression (Kountz et al, ). To increase membrane expression of this receptor, we used N‐terminally truncated receptors with additional mutations in the third intracellular loop and C‐tail to gain insights on the phosphorylated residues.…”

Novel Structural Approaches to Study GPCR Regulation

“…When expressed, it exhibits a predominant intracellular location [ 119 ], which seems be due to a domain located at the amino terminus [ 120 , 121 , 122 ]; therefore, amino terminus truncation is a suitable experimental procedure to achieve α 1D -adrenoceptor expression at the plasma membrane [ 96 , 114 , 120 , 121 , 122 ]. Interestingly, recent detailed work has shown, using receptor affinity purification and MS, that in multiple human cell lines, α 1D -adrenoceptors are expressed both as the full-length form and also as an amino terminus-truncated protein [ 123 ]. A cleavage site was identified at the L910/V91 site, and it was suggested that the proteolytic processing of the amino terminus is a physiological mechanism to achieve membrane location of α 1D -adrenoceptors with optimal functional properties [ 123 ].…”

“…Interestingly, recent detailed work has shown, using receptor affinity purification and MS, that in multiple human cell lines, α 1D -adrenoceptors are expressed both as the full-length form and also as an amino terminus-truncated protein [ 123 ]. A cleavage site was identified at the L910/V91 site, and it was suggested that the proteolytic processing of the amino terminus is a physiological mechanism to achieve membrane location of α 1D -adrenoceptors with optimal functional properties [ 123 ].…”