Regulation of G protein-coupled receptor trafficking and signaling by Rab GTPases

Abstract

Rab GTPases play an essential role in the regulation of intracellular transport including the budding, tethering, and fusion of vesicles as well as organelle motility. The regulation of G protein-coupled receptor (GPCR) trafficking by Rab GTPases has traditionally been regarded as a non-specific process that facilitates the movement of the receptors between intracellular membrane compartments. Thus, alterations in GPCR signal transduction and trafficking following the overexpression of constitutively active and dominant negative Rabs were originally considered to be solely the passive by-product of perturbations in intracellular compartmental dynamics. Recently, an explosion of experimental studies has provided increasingly convincing evidence that receptor trafficking actively affects the signal transduction of cargo proteins and that the signaling of GPCR vesicular cargo can in turn modulate Rab GTPase regulated intracellular transport processes. This research is revealing how different Rabs coordinate with themselves and other regulatory molecules to mediate protein trafficking, as well as uncovers novel functions for traditional Rabs, while illustrating the active role these trafficking molecules play in pathology of disease. Recently published in the Journal of Neuroscience, Esseltine et al., present a novel role for the typified exocytic small G protein Rab8 in the intracellular trafficking and signal transduction of metabotropic glutamate receptor 1.

Keywords: :

• G protein-coupled receptors
• Rab GTPase
• desensitization
• endocytosis
• glutamate
• signaling
• trafficking

This article refers to:

Role for Rab GTPases in the Regulation of GPCR Activity

Agonist-activation of signal transduction cascades through G protein-coupled receptors (GPCRs) comprises both G protein-dependent and -independent signaling resulting in the activation of parallel signaling cascades and complex signaling networks.¹ Because cells express hundreds of different receptors, a mechanism to organize signal cascades must be put in place. One major mechanism of spatiotemporal signal organization includes protein intracellular trafficking.² In addition to contributing to the rapid and effective desensitization of GPCR signaling, the endocytosis and intracellular trafficking of GPCRs can spatially and temporally determine G protein-dependent and -independent signaling pathways. Membrane trafficking may also provide novel compartments for signal transduction. Receptors, such as the β₂-adrenergic receptor (β₂AR), which recycle quickly and efficiently, also resensitize following either persistent or repeated agonist activation, whereas receptors, such as angiotensin type 1 receptor (AT1R), are retained in early endosomes and remain desensitized for longer periods of time.³ However, receptor trafficking can be much more complex, as β₂AR recycling has also been shown to actually switch the receptor’s traditional coupling with Gα_s to Gα_i, and the AT1R compartmentalizes extracellular regulated kinase (ERK1/2) complexes to endosomes.^1,4

Less well understood are the direct and indirect roles of GPCR signal transduction on the regulation of the trafficking machinery itself, although it is now proposed that receptors participate in the modulation of their own intracellular trafficking.^5,6 For example, AT1R activation causes GDP for GTP exchange on Rab5a resulting in Rab5 activation and homotypic endosomal vesicular fusion, while p38 MAPK activation stimulates the formation of Rab5-guanine dissociation inhibitor (GDI) complexes, thereby increasing endocytosis.^5,7 PKA activation by the β₂AR regulates Rab4, but not Rab11 recycling pathways, and β₂AR also modulates the geranyl-geranylation of both Rab11 and Rab8, altering the ability of these Rabs to associate with membranes, and thus modifying their overall functional activity.^8,9 Rab GTPases have been shown to bind to a number of additional GPCRs including the α_2BAR, thromboxane A2 receptor, prostacyclin receptor and metabotropic glutamate receptor 1a (mGluR1a).^5,10-14 The association of Rab GTPases with GPCRs is mediated by specific amino acid residues within the carboxyl-terminal tails of GPCRs that do not conform to a conserved consensus sequence. Rab binding to the AT1R C-tail is mediated by proline residue 354 and cysteine residue 355, and Rab4, Rab5, Rab7 and Rab11 each compete for the same binding site.¹⁵ The functional consequence of these competitive interactions determines whether the AT1R is retained in the early endosomal compartment, is dephosphorylated and recycled back to the plasma or is targeted to late endosomes for lysosomal degradation.^15,16 In contrast, Rab 11 binding to the thromboxane A2 receptor C-tail is mediated by amino acid residues 335–345, whereas Rab11 binding to the β₂AR C-tail is mediated a bipartite binding motif, with arginine 333 and lysine 348 representing the essential amino acid residues mediating Rab11 binding to the receptor.^10,11 The precise localization of Rab8 interactions with the mGluR1a C-tail has yet to be determined, but Rab8 does not regulate the activity of the mGluR1b alternative splice variant lacking an extended carboxyl-terminal tail.¹⁴

Rab8 Regulation of GPCR Activity

Rab8 is enriched in the brain, is localized to Golgi, vesicles and membrane ruffles, is involved in trafficking of basolateral proteins in polarized epithelial cells and plays a role in neurite outgrowth.¹⁷ Rab8 is also documented in the polarized transport of rhodopsin in photoreceptor cells and is found in the primary cilia of the cell.^18-20 Rab8 also directly associates with the α_2B- and β₂-adrenergic receptors, and a GDP-bound dominant negative Rab8 mutant blocks α_2BAR cell surface expression and ERK1/2 activation, but has no effect on β₂AR localization and signaling.¹² In a recent report, Esseltine and coworkers¹⁴ have demonstrated that Rab8 also interacts in an agonist regulated manner with the long carboxyl-terminal tail of the mGluR1a, but not mGluR1b splice variant. As a consequence, the association of Rab8 with mGluR1a selectively regulates mGluR1a endocytosis and signaling. Specifically, Rab8 functions to attenuate both mGluR1a internalization and inositol phosphate signaling in human embryonic kidney (HEK 293) cells and Ca²⁺ signaling in primary hippocampal neurons. Thus, contrary to other reports, which indicate that Rab8 expression facilitates receptor signaling by increased cell surface expression, Rab8-mediated increases in mGluR1a plasma membrane expression resulted in decreased receptor signaling both in HEK 293 cells and hippocampal neurons.

The attenuation of mGluR1a-stimulated inositol phosphate formation following Rab8 overexpression is reversed by the treatment of HEK 293 cells with the protein kinase C (PKC) inhibitors bisindolylmaleimide-1 and chelerythrine chloride.¹⁴ This suggests the Rab8 mediated attenuation of mGluR1a endocytosis may serve to increase second messenger-dependent kinase-mediated phosphorylation and desensitization by preventing mGluR1a dephosphorylation. The dephosphorylation of GPCRs, such as the β₂AR, is dependent upon agonist-stimulated receptor endocytosis to the endosomal compartment where the β₂AR is dephosphorylated as it transits from the Rab5 positive early endosomal compartment to the Rab4 positive recycling endosomal compartment.²¹ Although dephosphorylation of cAMP-dependent protein kinase phosphorylated β₂AR does not require endocytosis,²² it remains unknown whether the dephosphorylation of PKC phosphorylated mGluR1a either occurs at the cell surface or requires endocytosis to endosomes. Thus, we hypothesize that mGluR1a dephosphorylation does not occur at the cell surface, and Rab8-dependent cell surface mGluR1a retention prevents the dephosphorylation of PKC phosphorylated mGluR1a in the endosomal compartment. The extent of mGluR1a endocytosis in a given cell type is likely not regulated by Rab8 alone, but also involves a dynamically balanced association with other proteins that regulate the constitutive and agonist-stimulated internalization of mGluR1a, such as G protein-coupled receptor kinase 2, phospholipase D and Ral A.^23-26 Future investigation will be required to determine the functional dynamics underlying the overall regulation of mGluR1a endocytosis and its relative role in regulating the desensitization and resensitization of mGluR1a responsiveness.

Multiple Rabs have been shown to be involved in the regulation of both endocytosis and recycling of GPCRs.^5,10-14 However, to our knowledge, Rab8 represents the first example of a Rab GTPase that functions to attenuate the endocytosis and trafficking of a GPCR, thereby providing an additional avenue by which Rabs regulate GPCR activity and localization. One consequence of blocking mGluR1a internalization in HEK 293 cells is to increase the cell surface localization of the receptor. While this increase in cell surface expression did not have an overall effect on mGluR1a signaling in an overexpression system (HEK 293 cells) or Ca²⁺ signaling in neurons, it is unknown whether this might increase the localization of mGluR1a expression at the post-synaptic membrane in neurons to modulate the efficacy of synaptic transmission. For example, it has previously been demonstrated that Rab8 plays an important role in regulating the synaptic delivery of AMPA receptors during long-term potentiation and during constitutive receptor recycling.^27,28 Thus, it is reasonable to hypothesize that Rab8 may also regulate the localization and insertion of mGluR1a into the post synaptic density as an adaptive response increased synaptic activity.

Role of Rab Proteins in Huntington Disease

Huntington disease is a neurodegenerative disorder caused by an abnormal polyglutamine expansion in the N-terminus of the huntingtin (Hdh) protein, leading to cell dysfunction and neuronal death.^29,30 As is found for Rab8-mediated desensitization of mGluR1a signaling, the attenuation of mGluR5 signaling observed in HdhQ111/Q111 mice is also PKC-dependent.³¹ This PKC-dependent attenuation of mGluR5 signaling is neuroprotective, as PKC inhibitor treatment of neurons derived from HdhQ111/Q111 mice that are treated with the mGluR1/5 selective agonist DHPG exhibit increased DHPG-mediated cell death when compared with control neurons. However, neurons derived from HdhQ111/Q111 mice that lack mGluR5 expression (HdhQ111/Q111 mice crossed with mGluR5 knockout mice) are resistant to the neurotoxic effects of the combined treatment with DHPG and a PKC inhibitor.

The Rab8 effector protein, optineurin, also interacts with both mGluR1a and poly-glutamine expanded mutant huntingtin protein to antagonize mGluR1a-stimulated inositol phosphate formation.³² Optineurin colocalizes with huntingtin in the Golgi apparatus, where it participates with Rab8 to coordinate post-Golgi protein transport.³³ However, whether Rab8 also contributes to the post-Golgi transport of mGluR1a/5 and whether this is altered in mutant huntingtin mouse model remains to be determined. Another Rab GTPase, Rab11, likely also contributes to the pathology of Huntington disease. In particular, Rab11-dependent vesicle formation is impaired in fibroblasts derived in Huntington disease patients, and Rab11 dominant negative protein expressing adult mouse brains display a similar neurodegenerative phenotype to huntingtin mutant mouse models of Huntington disease.^34,35 Both Rab 8 and Rab11 associate with the actin motor protein myosin Vb to regulate trafficking.³⁶ Therefore, it is possible that Rab8 and Rab11 work in concert to regulate mGluR1a/5 protein trafficking under normal conditions and that this is altered in Huntington disease. In summary, while it remains unknown what the relationship Rab8/optineurin interactions have to the pathophysiology of Huntington’s disease, our observations suggest a potential link between Rab8, optineurin and mutant huntingtin protein in the regulation of mGluR1/5 activity in Huntington disease.

Acknowledgments

This work was supported by operating grants to S.S.G.F. from the Canadian Institutes of Health Research (CIHR) MOP 119437 and MOP 111093. J.L.E. was the recipient of an Ontario Graduate Studentship. S.S.G.F. holds a Tier I Canada Research Chair in Molecular Neurobiology and is a Career Investigator of the Heart and Stroke Foundation of Ontario.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.