title: Protein Kinase C Activation Promotes α1B-Adrenoceptor Internalization and Late Endosome Trafficking through Rab9 Interaction. Role in Heterologous Desensitization.
abstract: Upon agonist stimulation,α1B-adrenergic receptors couple to Gqproteins, calcium signaling and protein kinase C activation; subsequently, the receptors are phosphorylated, desensitized, and internalized. Internalization seems to involve scaffolding proteins, such asβ-arrestin and clathrin. However, the fine mechanisms that participate remain unsolved. The roles of protein kinase C and the small GTPase, Rab9, inα1B-AR vesicular traffic were investigated by studyingα1B-adrenergic receptor-Rab protein interactions, using Förster resonance energy transfer (FRET), confocal microscopy, and intracellular calcium quantitation. In human embryonic kidney 293 cells overexpressingDiscosomaspp. red fluorescent protein (DsRed)-taggedα1B-ARs and enhanced green fluorescent protein--tagged Rab proteins, pharmacological protein kinase C activation mimickedα1B-AR traffic elicited by nonrelated agents, such as sphingosine 1-phosphate (i.e., transientα1B-AR-Rab5 FRET signal followed by a sustainedα1B-AR-Rab9 interaction), suggesting brief receptor localization in early endosomes and transfer to late endosomes. This latter interaction was abrogated by blocking protein kinase C activity, resulting in receptor retention at the plasma membrane. Similar effects were observed when a dominant-negative Rab9 mutant (Rab9-GDP) was employed. Whenα1B-adrenergic receptors that had been mutated at protein kinase C phosphorylation sites (S396A, S402A) were used, phorbol ester-induced desensitization of the calcium response was markedly decreased; however, interaction with Rab9 was only partially decreased and internalization was observed in response to phorbol esters and sphingosine 1-phosphate. Finally, Rab9-GDP expression did not affect adrenergic-mediated calcium response but abolished receptor traffic and altered desensitization. Data suggest that protein kinase C modulatesα1B-adrenergic receptor transfer to late endosomes and that Rab9 regulates this process and participates in G protein-mediated signaling turn-off.
authors.affiliation: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad de México (M.A.A.-M., D.A.H.-E., G.C.-R., M.T.R.-A., J.A.G.-S.) and Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional-CINVESTAV, Col. San Pedro Zacatenco, Ciudad de México (G.R.-C.).
authors.forename: Marco A
authors.initials: MA
authors.lastname: Alfonzo-Méndez
authors.affiliation: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad de México (M.A.A.-M., D.A.H.-E., G.C.-R., M.T.R.-A., J.A.G.-S.) and Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional-CINVESTAV, Col. San Pedro Zacatenco, Ciudad de México (G.R.-C.).
authors.forename: David A
authors.initials: DA
authors.lastname: Hernández-Espinosa
authors.affiliation: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad de México (M.A.A.-M., D.A.H.-E., G.C.-R., M.T.R.-A., J.A.G.-S.) and Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional-CINVESTAV, Col. San Pedro Zacatenco, Ciudad de México (G.R.-C.).
authors.forename: Gabriel
authors.initials: G
authors.lastname: Carmona-Rosas
authors.affiliation: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad de México (M.A.A.-M., D.A.H.-E., G.C.-R., M.T.R.-A., J.A.G.-S.) and Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional-CINVESTAV, Col. San Pedro Zacatenco, Ciudad de México (G.R.-C.).
authors.forename: M Teresa
authors.initials: MT
authors.lastname: Romero-Ávila
authors.affiliation: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad de México (M.A.A.-M., D.A.H.-E., G.C.-R., M.T.R.-A., J.A.G.-S.) and Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional-CINVESTAV, Col. San Pedro Zacatenco, Ciudad de México (G.R.-C.).
authors.forename: Guadalupe
authors.initials: G
authors.lastname: Reyes-Cruz
authors.affiliation: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad de México (M.A.A.-M., D.A.H.-E., G.C.-R., M.T.R.-A., J.A.G.-S.) and Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional-CINVESTAV, Col. San Pedro Zacatenco, Ciudad de México (G.R.-C.) agarcia@ifc.unam.mx.
authors.forename: J Adolfo
authors.initials: JA
authors.lastname: García-Sáinz
mesh_terms: D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005453:Fluorescence; D020558:GTP Phosphohydrolases / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008954:Models, Biological; D009638:Norepinephrine / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism
chemical_list: D018340:Receptors, Adrenergic, alpha-1; D011493:Protein Kinase C; D020558:GTP Phosphohydrolases; C079856:RAB9A protein, human; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D009638:Norepinephrine