Protein Kinase C Activation Promotes α_1B-Adrenoceptor Internalization and Late Endosome Trafficking through Rab9 Interaction. Role in Heterologous Desensitization

Abstract

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

Heterologous phosphorylation–induced formation of a stability lock permits regulation of inactive receptors by β-arrestins

“…This may explain our finding that the PKC-induced β-arrestin2 recruitment directed the receptor to Rab7-enriched late endosomes to a lesser extent than AngII. Interestingly, the heterologous β-arrestin2 recruitment to α 1B-adrenergic receptor was shown to promote late endosomal trafficking, indicating that this type of regulation exhibits receptor specificity (59). Additionally, in a previous study we showed that the intracellular fate of AT1R is ligand-specific (48).…”

Updates in the function and regulation of α1‐adrenoceptors

“…Agonist‐activated α 1B ‐adrenoceptors (associated with homologous desensitization) mainly interacted with proteins present in early endosomes, such as early endosome antigen 1, Rab5, Rab4, and Rab11, but not with the late endosome markers Rab9 and Rab7 (Alfonzo‐Méndez et al, ; Castillo‐Badillo et al, ). In contrast, the activation of other receptors (such as sphingosine 1‐phosphate receptors) or direct activation of PKC by phorbol esters (heterologous desensitization) induced a brief α 1B ‐adrenoceptor–Rab5 interaction, a more pronounced and sustained one with Rab9, and some interaction with Rab7 (Alfonzo‐Méndez, Hernández‐Espinosa, et al, ; Castillo‐Badillo et al, ). It is worth mentioning that the action of sphingosine‐1‐phosphate on the phosphorylation/desensitization/internalization of α 1B ‐adrenoceptors mainly depended on the activation of PKC (Alfonzo‐Méndez, Hernández‐Espinosa, et al, ).…”

Free fatty acid receptor 4 agonists induce lysophosphatidic acid receptor 1 (LPA1) desensitization independent of LPA1 internalization and heterodimerization

“…Monoclonal anti‐GFP antibody (cat number 632381) was purchased from Clonthech, while secondary antibodies were from Zymed (Thermo Fisher Scientific) or Jackson ImmunoResearch Laboratories (West Grove, PA, USA) and chemiluminescence kits were from Millipore Corporation (Merck‐Millipore). Other materials were obtained from previously published sources .…”

“…HEK 293 cells (American Type Culture Collection, Manassas, VA, USA) were cultured as described . These cells do not appear to express functional free fatty acid receptors but do express LPA 1 receptors .…”

“…There is a wide range of examples of crosstalk among membrane receptors and one of the possible consequences of this is desensitization, in many cases associated with receptor phosphorylation and internalization . These processes have been shown for LPA 1 receptors . Another interesting event that can take place with GPCRs is their homo and heterodimerization .…”