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Abstract
Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs. heterologous).
Highlights
The G protein-coupled receptor (GPCR) superfamily comprises more than 600 distinct seven transmembrane-spanning members, and represents the largest group of integral membrane proteins [1,2,3,4]
We observed that wild type and DsRed-tagged α1B-adrenergic receptors are phosphoproteins whose phosphorylation state is increased by agents that induce desensitization such as noradrenaline or sphingosine 1-phosphate (Fig. 2, panel A)
Internalization was mainly evidenced by a decrease in the delineation of the plasma membrane by fluorescence and by an increase in the number and intensity of fluorescent vesicles, which is consistent with what has been observed with enhanced green fluorescent protein (EGFP)-tagged α1B-adrenergic receptors [42]
Summary
The G protein-coupled receptor (GPCR) superfamily comprises more than 600 distinct seven transmembrane-spanning members, and represents the largest group of integral membrane proteins [1,2,3,4]. These receptors mediate a plethora of processes, from sexual reproduction in unicellular eukaryotes, such as yeasts, to olfaction, vision, cognition, pain perception, and endocrine and exocrine secretion in vertebrates, and they participate in essentially all major physiological processes in mammals. Internalization and recycling are central parts of the regulation of GPCR signaling These processes take place under baseline conditions The best defined endocytic route used by GPCRs is the clathrin-mediated pathway [11, 12]
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