Protein Kinase C Activation Promotes α1B-Adrenoceptor Internalization and Late Endosome Trafficking through Rab9 Interaction. Role in Heterologous Desensitization

Upon agonist stimulation, alpha(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 beta-arrestin and clathrin. However, the fine mechanisms that participate remain unsolved. The roles of protein kinase C and the small GTPase, Rab9, in alpha(1B)-AR vesicular traffic were investigated by studying alpha(1B)-adrenergic receptor-Rab protein interactions, using Forster resonance energy transfer (FRET), confocal microscopy, and intracellular calcium quantitation. In human embryonic kidney 293 cells overexpressing Discosoma spp. red fluorescent protein (DsRed)-tagged alpha(1B)-ARs and enhanced green fluorescent protein- tagged Rab proteins, pharmacological protein kinase C activationmimicked a1B-AR traffic elicited by nonrelated agents, such as sphingosine 1-phosphate (i.e., transient alpha(1B)-AR-Rab5 FRET signal followed by a sustained a1B-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 a1B-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 alpha(1B)-adrenergic receptor transfer to late endosomes and that Rab9 regulates this process and participates in G protein-mediated signaling turn-off.