Alfonzo-Méndez Marco A, Hernández-Espinosa David A, Carmona-Rosas Gabriel, Romero-Ávila M Teresa, Reyes-Cruz Guadalupe, García-Sáinz J Adolfo
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.).
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.)
Mol Pharmacol. 2017 Apr;91(4):296-306. doi: 10.1124/mol.116.106583. Epub 2017 Jan 12.
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.
在激动剂刺激下，β - 肾上腺素能受体与G蛋白、钙信号传导和蛋白激酶C激活偶联；随后，受体被磷酸化、脱敏并内化。内化似乎涉及支架蛋白，如β - 抑制蛋白和网格蛋白。然而，参与其中的精细机制仍未解决。通过使用荧光共振能量转移（FRET）、共聚焦显微镜和细胞内钙定量研究β - 肾上腺素能受体 - Rab蛋白相互作用，研究了蛋白激酶C和小GTP酶Rab9在β - AR囊泡运输中的作用。在过表达带有红色荧光蛋白（DsRed）标签的β - ARs和增强型绿色荧光蛋白标记的Rab蛋白的人胚肾293细胞中，药理学上的蛋白激酶C激活模拟了由无关试剂（如1 - 磷酸鞘氨醇）引发的β - AR运输（即短暂的β - AR - Rab5 FRET信号，随后是持续的β - AR - Rab9相互作用），这表明受体在早期内体中短暂定位并转移至晚期内体。通过阻断蛋白激酶C活性可消除后者的相互作用，导致受体保留在质膜上。当使用显性负性Rab9突变体（Rab9 - GDP）时，也观察到了类似的效果。当使用在蛋白激酶C磷酸化位点（S396A、S402A）发生突变的β - 肾上腺素能受体时，佛波酯诱导的钙反应脱敏明显降低；然而，与Rab9的相互作用仅部分降低，并且观察到对佛波酯和1 - 磷酸鞘氨醇有内化反应。最后，Rab9 - GDP的表达不影响肾上腺素能介导的钙反应，但消除了受体运输并改变了脱敏作用。数据表明蛋白激酶C调节β - 肾上腺素能受体向晚期内体的转移，并且Rab9调节这一过程并参与G蛋白介导的信号关闭。
