Signals from a Sunken Ship

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Science Signaling  25 Aug 2009:
Vol. 2, Issue 85, pp. ec281
DOI: 10.1126/scisignal.285ec281

G protein–coupled receptors (GPCRs) at the plasma membrane bind to their ligands and couple to heterotrimeric G proteins, leading to the generation of second messengers such as cyclic adenosine monophosphate (cAMP). Phosphorylation of activated GPCRs leads to their desensitization and internalization, which is thought to result in signal termination, although β-arrestin–dependent signaling can also be initiated. However, aspects of the spatial and temporal regulation of GPCRs are still unclear. Calebiro et al. generated transgenic mice expressing a fluorescent resonance energy transfer–based sensor of cAMP and used fluorescence microscopy to examine signaling by the thyroid-stimulating hormone (TSH) receptor, a GPCR, in thyroid follicles in 3D culture. Although short-term stimulation with TSH led to transient cAMP generation, prolonged stimulation or stimulation with higher concentrations of TSH led to the accumulation of cAMP. Confocal microscopy experiments performed with a fluorescently tagged TSH showed internalization of the TSH receptor, after which it partially colocalized with Gαs and adenylyl cyclase type III at intracellular vesicles. Blocking the internalization of the TSH receptor abolished accumulation of cAMP. Subcellular fractionation and in vitro adenylyl cyclase assays showed that the intracellular fractions of thyroid cells responded to TSH by generating cAMP. TSH-stimulated depolymerization of actin was inhibited in thyroid cells treated with an endocytosis inhibitor compared with that in control cells, and this inhibition correlated with the decreased TSH-dependent phosphorylation of VASP, a regulator of actin polymerization. Together, these data suggest not only that internalized TSH receptors continue to generate cAMP signals as they did at the membrane but also that the subcellular localization of these receptors may lead to qualitative differences in signaling outcomes.

D. Calebiro, V. O. Nikolaev, M. C. Gagliani, T. de Filippis, C. Dees, C. Tacchetti, L. Persani, M. J. Lohse, Persistent cAMP-signals triggered by internalized G-protein–coupled receptors. PLoS Biol. 7, e1000172 (2009). [PubMed]

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