The adaptor complex 2 directly interacts with the alpha 1b-adrenergic receptor and plays a role in receptor endocytosis.

Using the yeast two-hybrid system, we identified the mu 2 subunit of the clathrin adaptor complex 2 as a protein interacting with the C-tail of the alpha 1b-adrenergic receptor (AR). Direct association between the alpha 1b-AR and mu 2 was demonstrated using a solid phase overlay assay. The alpha 1b-AR/mu 2 interaction occurred inside the cells, as shown by the finding that the transfected alpha 1b-AR and the endogenous mu 2 could be coimmunoprecipitated from HEK-293 cell extracts. Mutational analysis of the alpha 1b-AR revealed that the binding site for mu 2 does not involve canonical YXX Phi or dileucine motifs but a stretch of eight arginines on the receptor C-tail. The binding domain of mu 2 for the receptor C-tail involves both its N terminus and the subdomain B of its C-terminal portion. The alpha 1b-AR specifically interacted with mu 2, but not with the mu 1, mu 3, or mu 4 subunits belonging to other AP complexes. The deletion of the mu 2 binding site in the C-tail markedly decreased agonist-induced receptor internalization as demonstrated by confocal microscopy as well as by the results of a surface receptor biotinylation assay. The direct association of the adaptor complex 2 with a G protein-coupled receptor has not been reported so far and might represent a common mechanism underlying clathrin-mediated receptor endocytosis.

Sox4 participates in the modulation of Schwann cell myelination.

In order to identify new regulators of Schwann cell myelination potentially playing a role in peripheral nervous system (PNS) pathologies, we analysed gene expression profiling data from three mouse models of demyelinating neuropathies and from the developing PNS. This analysis revealed that Sox4, which encodes a member of the Sry-related high-mobility group box protein family, was consistently upregulated in all three analysed models of neuropathy. Moreover, Sox4 showed a peak in its expression during development that corresponded with the onset of myelination. To gain further insights into the role of Sox4 in PNS development, we generated a transgenic mouse that specifically overexpresses Sox4 in Schwann cells. Sox4 overexpression led to a temporary delay in PNS myelination without affecting axonal sorting. Importantly, we observed that, whereas Sox4 mRNA could be efficiently overexpressed, Sox4 protein expression in Schwann cells was strictly regulated. Finally, our data showed that enforced expression of Sox4 in the mouse model for Charcot-Marie-Tooth 4C aggravated its neuropathic phenotype. Together, these observations reveal that Sox4 contributes to the regulation of Schwann cell myelination, and also indicates its involvement in the pathophysiology of peripheral neuropathies.

Molecular mechanism of a green-shifted, pH-dependent red fluorescent protein mKate variant.

Fluorescent proteins that can switch between distinct colors have contributed significantly to modern biomedical imaging technologies and molecular cell biology. Here we report the identification and biochemical analysis of a green-shifted red fluorescent protein variant GmKate, produced by the introduction of two mutations into mKate. Although the mutations decrease the overall brightness of the protein, GmKate is subject to pH-dependent, reversible green-to-red color conversion. At physiological pH, GmKate absorbs blue light (445 nm) and emits green fluorescence (525 nm). At pH above 9.0, GmKate absorbs 598 nm light and emits 646 nm, far-red fluorescence, similar to its sequence homolog mNeptune. Based on optical spectra and crystal structures of GmKate in its green and red states, the reversible color transition is attributed to the different protonation states of the cis-chromophore, an interpretation that was confirmed by quantum chemical calculations. Crystal structures reveal potential hydrogen bond networks around the chromophore that may facilitate the protonation switch, and indicate a molecular basis for the unusual bathochromic shift observed at high pH. This study provides mechanistic insights into the color tuning of mKate variants, which may aid the development of green-to-red color-convertible fluorescent sensors, and suggests GmKate as a prototype of genetically encoded pH sensors for biological studies.