title: Molecular determinants of the human α2C-adrenergic receptor temperature-sensitive intracellular traffic.
abstract: The human α2C-adrenergic receptor (α2C-AR) is localized intracellularly at physiologic temperature. Decreasing the environmental temperature strongly stimulates the receptor transport to the cell surface. In contrast, rat and mouse α2C-AR plasma membrane levels are less sensitive to decrease in temperature, whereas the opossum α2C-AR cell surface levels are not changed in these conditions. Structural analysis demonstrated that human α2C-AR has a high number of arginine residues in the third intracellular loop and in the C-terminus, organized as putative RXR motifs. Although these motifs do not affect the receptor subcellular localization at 37°C, deletion of the arginine clusters significantly enhanced receptor plasma membrane levels at reduced temperature. We found that this exaggerated transport of the human receptor is mediated by two functional arginine clusters, one in the third intracellular loop and one in the C-terminus. This effect is mediated by interactions with COPI vesicles, but not by 14-3-3 proteins. In rat α2C-AR, the arginine cluster from the third intracellular loop is shifted to the left due to three missing residues. Reinsertion of these residues in the rat α2C-AR restored the same temperature sensitivity as in the human receptor. Proteomic and coimmunoprecipitation experiments identified pontin as a molecule having stronger interactions with human α2C-AR compared with rat α2C-AR. Inhibition of pontin activity enhanced human receptor plasma membrane levels and signaling at 37°C. Our results demonstrate that human α2C-AR has a unique temperature-sensitive traffic pattern within the G protein-coupled receptor class due to interactions with different molecular chaperones, mediated in part by strict spatial localization of specific arginine residues.
authors: Department of Pharmacology, College of Medicine, Howard University, Washington, DC (C.M.F.); Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana (A.K.P., J.J.G.); Department of Cancer Biology, Wake Forest University School of Medicine, Winston Salem, North Carolina (A.K.P.); and Louisiana State University Health Sciences Center Proteomics Core Facility, New Orleans, Louisiana (J.J.G.) catalin.filipeanu@howard.edu.
authors: Catalin M
authors: CM
authors: Filipeanu
authors: Department of Pharmacology, College of Medicine, Howard University, Washington, DC (C.M.F.); Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana (A.K.P., J.J.G.); Department of Cancer Biology, Wake Forest University School of Medicine, Winston Salem, North Carolina (A.K.P.); and Louisiana State University Health Sciences Center Proteomics Core Facility, New Orleans, Louisiana (J.J.G.).
authors: Ashok K
authors: AK
authors: Pullikuth
authors: Department of Pharmacology, College of Medicine, Howard University, Washington, DC (C.M.F.); Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana (A.K.P., J.J.G.); Department of Cancer Biology, Wake Forest University School of Medicine, Winston Salem, North Carolina (A.K.P.); and Louisiana State University Health Sciences Center Proteomics Core Facility, New Orleans, Louisiana (J.J.G.).
authors: Jessie J
authors: JJ
authors: Guidry
mesh_terms: D000818:Animals; D001120:Arginine / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D021381:Protein Transport / Q000502:physiology*; D040901:Proteomics / Q000379:methods; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013696:Temperature
chemical_list: D018341:Receptors, Adrenergic, alpha-2; D043562:Receptors, G-Protein-Coupled; D001120:Arginine