Arrestin Is Required for Agonist-induced Trafficking of Voltage-dependent Calcium Channels

Akil Puckerin,Lanying Liu,Natasha Permaul,Paul Carman,Jessica Lee,Maria A Diverse-Pierluissi

doi:10.1074/jbc.m605000200

Akil Puckerin, Lanying Liu + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m605000200

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Abstract

Many metabotropic receptors in the nervous system act through signaling pathways that result in the inhibition of voltage-dependent calcium channels. Our previous findings showed that activation of seven-transmembrane receptors results in the internalization of calcium channels. This internalization takes place within a few seconds, raising the question of whether the endocytic machinery is in close proximity to the calcium channel to cause such rapid internalization. Here we show that voltage-dependent calcium channels are pre-associated with arrestin, a protein known to play a role in receptor trafficking. Upon GABAB receptor activation, receptors are recruited to the arrestin-channel complex and internalized. beta-Arrestin 1 selectively binds to the SNARE-binding region of the calcium channel. Peptides containing the arrestin-binding site of the channel disrupt agonist-induced channel internalization. Taken together these data suggest a novel neuronal role for arrestin.

Highlights

Many metabotropic receptors in the nervous system act number of voltage-dependent calcium channels at the memthrough signaling pathways that result in the inhibition of brane
We demonstrate that ␤-arrestin 1 is associated with tion of whether the endocytic machinery is in close proximity Cav2.2 channels and that activation of 7TMRs results in the to the calcium channel to cause such rapid internalization. formation of an arrestin-receptor-channel complex
E ay channel, 2 ␮g of His6-tagged recombinant protein containing R M the channel sequence was mixed and incubated with 2 ␮g of chick dorsal root ganglion (DRG) neurons treated with saline or baclofen (Fig. 2a) giving further support to the observation that arrestin is preassociated with the calcium channel in an agonist-independent fashion

Summary

EXPERIMENTAL PROCEDURES

Materials—The following primary antibodies were used in these studies: rabbit anti-pan-␣1 (1:200,1.5 ␮g/ml) (Alomone Labs, Jerusalem, Israel), anti-arrestin (1:500, BD Biosciences), and anti-GABAR1 (1:200, Chemicon). For the experiments presented in this report, cal- slices; in the middle slices the fluorescence signal forms a ring cium current has been corrected for rundown by measuring around the periphery of the cell suggesting association with the calcium current as a function of time in control cells without membrane The Pearson correlation coefficient between calcium chanment, DRG neurons were lysed with ice-cold buffer nels and arrestin is 0.73 Ϯ 0.09 in saline-treated cells and 0.8 Ϯ (phosphate-buffered saline, pH 7.4, containing 250 ␮M sodium 0.06 in baclofen-treated cells (n ϭ 25) Together these results pervanadate, 1% (v/v) Nonidet P-40, 1 mM Pefabloc, 1 mM suggest that arrestin and Cav2.2 channels are preassociated in EDTA, 1 mM EGTA, 10 ␮g/ml pepstatin, 10 ␮g/ml leupeptin, the cell surface and are internalized upon 7TMR activation. Immunodetection of arrestin was carried out using anti-arrestin (Chemicon, 1:1000)

To test interactions of recombinant arrestin with the calcium

DISCUSSION

Findings

ADDITIONS AND CORRECTIONS