α2B-Adrenergic Receptor Interaction with Tubulin Controls Its Transport from the Endoplasmic Reticulum to the Cell Surface

Abstract

It is well recognized that the C terminus (CT) plays a crucial role in modulating G protein-coupled receptor (GPCR) transport from the endoplasmic reticulum (ER) to the cell surface. However the molecular mechanisms that govern CT-dependent ER export remain elusive. To address this issue, we used ␣ 2B -adrenergic receptor (␣ 2B -AR) as a model GPCR to search for proteins interacting with the CT. By using peptide-conjugated affinity matrix combined with proteomics and glutathione S-transferase fusion protein pull-down assays, we identified tubulin directly interacting with the ␣ 2B -AR CT. The interaction domains were mapped to the acidic CT of tubulin and the basic Arg residues in the ␣ 2B -AR CT, particularly Arg-437, Arg-441, and Arg-446. More importantly, mutation of these Arg residues to disrupt tubulin interaction markedly inhibited ␣ 2B -AR transport to the cell surface and strongly arrested the receptor in the ER. These data provide the first evidence indicating that the ␣ 2B -AR C-terminal Arg cluster mediates its association with tubulin to coordinate its ER-to-cell surface traffic and suggest a novel mechanism of GPCR export through physical contact with microtubules.

The mechanism and function of mitogen-activated protein kinase activation by ARF1

“…[ 3 H]-RX821002 (specific activity = 50 Ci/mmol) was from Perkin Elmer Life Sciences. All other materials were obtained as described elsewhere [29-31]. …”

Regulation of α2B-Adrenergic Receptor-mediated Extracellular Signal-regulated Kinase 1/2 (ERK1/2) Activation by ADP-ribosylation Factor 1

“…Flow Cytometric Analysis of Receptor Expression-␣ 2B -AR expression at the cell surface was also measured by flow cytometry as described previously (29,33). HEK293 cells transfected with HA-␣ 2B -AR were suspended in PBS containing 1% FBS at a density of 4 ϫ 10 6 cells/ml and incubated with high affinity anti-HA-fluorescein (3F10) at a final concentration of 2 g/ml for 30 min at 4°C.…”

“…The radioligand [ 3 H]RX821002 (specific activity ϭ 41 Ci/mmol) was purchased from PerkinElmer Life Sciences. All other materials were obtained as described elsewhere (27)(28)(29).…”

Looped Host Defense Peptide CLP-19 Binds to Microtubules and Inhibits Surface Expression of TLR4 on Mouse Macrophages

“…TLR4, the principal signaling receptor for LPS, is synthesized, stored, and modified in the ER and Golgi apparatus system (37). The precise downstream effects and duration of activated TLR4 signaling relies on the highly regulated intracellular trafficking processes, including ER-to-Golgi-to-cell surface transport of the nascent receptors, agonist-induced cell surface-to-endosome internalization of the receptors, endosome-to-cell surface recycling of the internalized receptors, and intracellular targeting of the receptor to the lysosome for degradation (38). In each of these processes, microtubule-dependent vesicular transport plays an essential role, allowing rapid shuttling of cargo carriers to their correct destination (37,39,40).…”