The Intracellular Trafficking of the G Protein-coupled Receptor TPβ Depends on a Direct Interaction with Rab11

Abstract

Intracellular trafficking pathways of cell surface receptors following their internalization are the subject of intense research efforts. However, the mechanisms by which they recycle back to the cell surface are still poorly defined. We have recently demonstrated that the small Rab11 GTPase protein is a determinant factor in controlling the recycling to the cell surface of the ␤-isoform of the thromboxane A 2 receptor (TP␤) following its internalization. Here, we demonstrate with co-immunoprecipitation studies in HEK293 cells that there is a Rab11-TP␤ association occurring in the absence of agonist, which is not modulated by stimulation of TP␤. We show with purified TP␤ intracellular domains fused to GST and HISRab11 proteins that Rab11 interacts directly with the first intracellular loop and the C-tail of TP␤. Amino acids 335-344 of the TP␤ C-tail were determined to be essential for the interaction of Rab11 with this receptor domain. This identified sequence appears to be important in directing the intracellular trafficking of the receptor from the Rab5-positive intracellular compartment to the perinuclear recycling endosome. Interestingly, our data indicate that TP␤ interacts with the GDP-bound form, and not the GTP-bound form, of Rab11 which is necessary for recycling of the receptor back to the cell surface. To our knowledge, this is the first demonstration of a direct interaction between Rab11 and a transmembrane receptor.

Regulation of β2-Adrenergic Receptor Maturation and Anterograde Trafficking by an Interaction with Rab Geranylgeranyltransferase

“…Early experiments indicated that membrane translocation of Rab proteins preceded nucleotide exchange and activation (52,53). This is particularly interesting because we previously reported that GPCRs such as the ␤ 2 AR and TP␤ interact with Rab11a-GDP but not with Rab11a-GTP (22,23). It is also remarkable that the nongeranylgeranylated forms of Rab6a, Rab8a, and Rab11a, as well as the Rab11a-CCSS mutant were co-immunoprecipitated with the ␤ 2 AR.…”

“…We and others reported that an interaction between Rab proteins and GPCRs dictates the trafficking of the receptors (22)(23)(24)(25)(26)(27)(28)(29). However, whether other components of the Rab protein modification machinery or Rab effectors also interact with GPCRs remains to be determined.…”

“…Rab-mediated vesicular transport is well known to regulate membrane receptor trafficking, but less is known about whether membrane receptors conversely regulate the Rab-trafficking machinery. We and others showed that GPCRs interact with Rabs resulting in the regulation of receptor trafficking (22)(23)(24)(25)(26)(27)(28)(29). The task of unraveling whether membrane receptors interact with other elements of the Rab-associated machinery to direct their own trafficking remains.…”

“…We recently showed that an interaction between GPCRs and Rab11a directed receptor trafficking (22,23,32). A higher molecular mass form of Rab11a was detected after longer exposure of Western blot membranes, and this form was increased when GPCRs were co-expressed in HEK293 cells.…”

Heterodimerization of the α and β isoforms of the human thromboxane receptor enhances isoprostane signaling

“…Both TPα and TPβ are coupled primarily to phospholipase C, while the former may activate and the latter inhibit, adenylyl cyclase (AC) activity [7]. Additionally, dissimilar patterns of constitutive and agonist-dependent internalization of the membrane expressed TPα and TPβ have been observed, reportedly because of their differential interactions with regulatory proteins Rab11 and arrestin [8,9]. However despite advances elucidating their individual functional and regulatory characteristics, the distinction between TPα and TPβ with respect to their ultimate physiological or pathophysiological roles remains unclear.…”