Recycling of the human prostacyclin receptor is regulated through a direct interaction with Rab11a GTPase

Abstract

Running Title: Role of Rab11 and Rab4 in recycling of the human prostacyclin receptor.Key Words: human prostacyclin receptor, internalization, Rab11a, Rab4a, yeast-two-hybrid, GPCR. -2 - ABSTRACTThe human prostacyclin receptor (hIP) undergoes agonist-induced internalization but the mechanisms regulating its intracellular trafficking and/or recycling to the plasma membrane are poorly understood.Herein, we conducted a yeast-two-hybrid screen to identify proteins interacting with the carboxyl terminal (C)-tail domain of the hIP and discovered a novel interaction with Rab11a. This interaction was confirmed by co-immunoprecipitations in mammalian HEK293 and was augmented by cicaproststimulation. The hIP co-localized to Rab11-containing recycling endosomes in both HEK293 and endothelial EA.hy 926 cells in a time-dependent manner following cicaprost-stimulation. Moreover, over-expression of Rab11a significantly increased recycling of the hIP, while the dominant negative Rab11S25N impaired that recycling. Conversely, while the hIP co-localized to Rab4-positive endosomes in response to cicaprost, ectopic expression of Rab4a did not substantially affect overall recycling nor did Rab4a directly interact with the hIP. The specific interaction between the hIP and Rab11a was dependent on a 22 amino acid (Val 299 -Gln 320 ) sequence within its C-tail domain and was independent of isoprenylation of the hIP. This study elucidates a critical role for Rab11a in regulating trafficking of the hIP and has identified a novel Rab11 binding-domain (RBD) within its C-tail domain that is both necessary and sufficient to mediate interaction with Rab11a.

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

“…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.…”

New insights in the regulation of Rab GTPases by G protein-coupled receptors

“…20 We and others established that a direct interaction between a GPCR and a Rab GTPase appears to be necessary in the proper trafficking of the receptor. 4,7,12,15,[17][18][19] Rab-mediated vesicular transport is well known to regulate membrane receptor trafficking, but less is understood about whether membrane receptors conversely regulate the Rab-trafficking machinery. [21][22][23] In this regard, an elegant study performed by Seachrist et al determined that activation of the angiotensin type 1 receptor stimulates the GTP-loading of Rab5a, suggesting that the receptor could act as a potential GEF or might recruit a GEF protein, thereby increasing the level of GTP-bound Rab5a.…”

“…2 During the last decade, many studies described the involvement of Rab GTPases in the regulation of GPCR trafficking and signaling. Rab GTPases were shown to be key regulators of GPCR anterograde and retrograde transport (Rab1, Rab2, Rab6, and Rab8), [3][4][5][6] endocytosis (Rab5), [7][8][9][10][11] recycling (Rab4 and Rab11), [11][12][13][14][15][16][17][18][19] and degradation (Rab7). 20 We and others established that a direct interaction between a GPCR and a Rab GTPase appears to be necessary in the proper trafficking of the receptor.…”

Interaction of angio-associated migratory cell protein with the TPα and TPβ isoforms of the human thromboxane A2 receptor

“…These interactions mediate diverse functions such as the regulation of T lymphocyte migration through interaction of the CCR5 and CXCR4 chemokine receptors with myosin heavy chain IIA [46] and the regulation of the recycling of the prostacyclin receptor through a direct interaction with Rab11a [47,48]. In many cases, the intracellular carboxyl-terminal (C) tail of the GPCR has emerged as the critical binding domain for such interactions with various GIPs, largely due to its divergent sequence, structure and capacity to contain functionally distinct binding motifs [44,45,49].…”