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Sci. Signal., 16 October 2012
Vol. 5, Issue 246, p. ec266
[DOI: 10.1126/scisignal.2003690]


Plant Biology Internalization as Activation

Ernesto Andrianantoandro

Science Signaling, AAAS, Washington, DC 20005, USA

Signaling by G protein–coupled receptors (GPCRs) is different in animal and plant cells. Whereas in animal cells, GPCRs activate G proteins, in plants, the G protein is constitutively active and the GPCR functions as an inhibitor of the G protein. Arabadopsis has a noncanonical GPCR, AtRGS1, which contains a seven-transmembrane domain and a regulatory domain (RGS) and is required for glucose sensing, cell proliferation and elongation, and development. The Gα subunit (AtGPA1) is self-activating and is inhibited by the RGS of AtRGS1. Urano et al. examined the activity and localization of AtRGS1 in the presence of glucose. Microscopy revealed that fluorescent protein fusions of AtRGS1 and AtGPA1 were internalized and localized to the plasma membrane, respectively, in hypocotyl epidermal cells in response to glucose. AtRGS1 colocalized with an endosomal marker. Deletion of both AtGPA1 and the β subunit (AGB1), but not individual subunits, abrogated internalization of AtRGS1. Overexpression of AtGPA1Q222L, a constitutively active mutant deficient in GTP hydrolysis, triggered internalization in the absence of glucose, but deleting AGB1 inhibited the effect. AtRGS1E320K, a mutant that does not interact with AtGPA1, did not internalize in response to glucose or when coexpressed with AtGPA1Q222L, indicating that the receptor and the G protein needed to interact for internalization to occur. Because many GPCRs are regulated by phosphorylation, the authors searched for candidate kinases from among interaction partners previously identified in yeast complementation screens. Glucose failed to elicit AtRGS1 internalization in cells lacking the candidate kinases AtWNK8 or -10. Coimmunoprecipitation and in vitro phosphorylation studies showed that AtWNK8 phosphorylated AtRGS1. A triple point mutant lacking the sites phosphorylated by AtWNK8 did not internalize. Purifed AtWNK8 coimmunoprecipitated with purified AGB1 but not AtGPA1. AtWNK8 and AtRGS1 fused to fluorescent proteins associated at the plasma membrane in response to glucose. Expression of genes downstream of AtRGS1 signaling was decreased in cells lacking AtWNK8 or -10 and abrogated in cells lacking both kinases. These results suggest a mechanism for regulation of GPCRs in plants by receptor endocytosis: In the absence of glucose, self-activated Gα inhibited by and bound to the RGS of AtRGS1 is in equilibrium with inactive Gα bound to the β{gamma} subunit; glucose stabilizes the RGS-bound state, allowing free β{gamma} to recruit AtWNK8, which phosphorylates the RGS, thus promoting internalization of AtRGS1, leaving active Gα at the plasma membrane.

D. Urano, N. Phan, J. C. Jones, J. Yang, J. Huang, J. Grigston, J. P. Taylor, A. M. Jones, Endocytosis of the seven-transmembrane RGS1 protein activates G-protein-coupled signalling in Arabidopsis. Nat. Cell Biol. 14, 1079–1088 (2012). [PubMed]

Citation: E. Andrianantoandro, Internalization as Activation. Sci. Signal. 5, ec266 (2012).

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