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 ...
Pseudokinases and pseudophosphatases possess the ability to bind substrates without catalyzing their modification, thereby providing a mechanism to recruit potential phosphotargets away from active enzymes. Since many of these pseudoenzymes possess other characteristics such as localization signals, separate catalytic sites, and protein–protein interaction domains, they have the capacity to influence signaling dynamics in local environments. In a similar manner, the targeting of signaling enzymes to subcellular locations by A-kinase-anchoring proteins (AKAPs) allows for precise and local control of second messenger signaling events. Here, we will discuss how pseudoenzymes form ‘pseudoscaffolds’ and compare and contrast this compartment-specific regulatory role with the signal organization properties of AKAPs. The mitochondria will be the focus of this review, as they are dynamic organelles that influence a broad range of cellular processes such as metabolism, ATP synthesis, and apoptosis.
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