Publication Date:
2017-02-26
Description:
Upon agonist stimulation, α 1B -adrenergic receptors couple to G q proteins, calcium signaling and protein kinase C activation; subsequently, the receptors are phosphorylated, desensitized, and internalized. Internalization seems to involve scaffolding proteins, such as β -arrestin and clathrin. However, the fine mechanisms that participate remain unsolved. The roles of protein kinase C and the small GTPase, Rab9, in α 1B -AR vesicular traffic were investigated by studying α 1B -adrenergic receptor–Rab protein interactions, using Förster resonance energy transfer (FRET), confocal microscopy, and intracellular calcium quantitation. In human embryonic kidney 293 cells overexpressing Discosoma spp. red fluorescent protein (DsRed)-tagged α 1B -ARs and enhanced green fluorescent protein–-tagged Rab proteins, pharmacological protein kinase C activation mimicked α 1B -AR traffic elicited by nonrelated agents, such as sphingosine 1-phosphate (i.e., transient α 1B -AR-Rab5 FRET signal followed by a sustained α 1B -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 α 1B -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 modulates α 1B -adrenergic receptor transfer to late endosomes and that Rab9 regulates this process and participates in G protein-mediated signaling turn-off.
Print ISSN:
0026-895X
Electronic ISSN:
1521-0111
Topics:
Chemistry and Pharmacology
,
Medicine
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