RT Journal Article SR Electronic T1 Protein kinase C activation promotes α1B-adrenoceptor internalization and late endosome trafficking through Rab9 interaction. Role in heterologous desensitization JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP mol.116.106583 DO 10.1124/mol.116.106583 A1 Marco A. Alfonzo-Mendez A1 David A Hernandez-Espinosa A1 Gabriel Carmona-Rosas A1 M. Teresa Romero-Avila A1 Guadalupe Reyes-Cruz A1 J. Adolfo Garcia-Sainz YR 2017 UL http://molpharm.aspetjournals.org/content/early/2017/01/11/mol.116.106583.abstract AB Upon agonist stimulation, α1B-adrenergic receptors (α1B-ARs) couple to Gq, calcium signaling and protein kinase C activation; subsequently, the receptors are phosphorylated, desensitized and internalized. Internalization seems to involve scaffolding proteins, such as α-arrestin and clathrin. However, the fine mechanisms that participate remain unsolved. The roles of protein kinase C and the small GTPase, Rab9, in α1B-AR vesicular traffic were investigated by studying α1B-adrenergic receptor-Rab protein interactions, using Forster resonance energy transfer (FRET), confocal microscopy, and intracellular calcium quantitation. In HEK293 cells overexpressing DsRed-tagged α1B-ARs and EGFP-tagged Rab proteins, pharmacological protein kinase C activation mimicked α1B-AR traffic elicited by non-related agents, such as sphingosine 1-phosphate, i.e., transient α1B-AR-Rab5 FRET signal followed by a sustained α1B-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. When α1B-adrenergic receptors 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 modulates α1B-adrenergic receptor transfer to late endosomes and that Rab9 regulates this process and participates in G protein-mediated signaling turn-off.