Role of Rab GTPases in the export trafficking of G protein-coupled receptors

ABSTRACT

G protein-coupled receptors (GPCRs) constitute a superfamily of cell surface receptors that regulate a variety of cell functions. As the cell surface is the functional destination for most GPCRs, the cell surface targeting process represents a crucial checkpoint in controlling the functionality of the receptors. However, the molecular mechanisms underlying the cell surface delivery of newly synthesized GPCRs remain poorly understood. In this review, we will highlight the role of Rab GTPases in GPCR cell surface transport, particularly post-Golgi traffic, and discuss the underlying molecular mechanisms.

KEYWORDS:

• cell surface expression
• export trafficking
• G protein-coupled receptor
• post-Golgi traffic
• Rab26
• Rab8
• Rab GTPase

Regulation of intracellular trafficking of G protein-coupled receptors by Rab GTPases

G protein-coupled receptors (GPCRs) are the largest and the most structurally diverse family of signaling proteins.^1,2 They regulate a variety of cell functions and are the actual targets of about 30% of drugs on the market to treat human diseases. The precise function of GPCRs is tightly controlled by their appropriate intracellular trafficking, such as ER-Golgi-cell surface export, internalization, recycling and lysosome transport, which dictate the receptors to their functional destinations. After being synthesized and properly assembled in the endoplasmic reticulum (ER), GPCRs are transported forward in vesicles to the cell surface through the Golgi cisternae and the trans-Golgi network (TGN). Once at the cell surface, the receptors are able to bind to their ligands or drugs to produce physiologic or pharmacological responses. Ligand binding to the receptors may induce receptor internalization during which the receptors are moved from the cell surface to the endosomal compartment. Internalized receptors in endosomes can be sorted to the recycling, degradation or retrograde pathways which transport the receptors to the cell surface, lysosomes and the TGN, respectively. Over the past decades, most studies on GPCR trafficking have focused on internalization, recycling and degradation pathways.^3-5 However, the molecular mechanisms underlying the anterograde transport of nascent GPCRs en route from the ER through the Golgi to the cell surface remain poorly understood.

Rab GTPases are the largest branch of the Ras-related GTPase superfamily, consisting of more than 60 members, and are involved in almost every step of vesicle-mediated endocytosis and exocytosis. Each Rab GTPase has a distinct subcellular localization pattern that correlates with its function in directing cargo transport between specific subcellular compartments. As with all GTPases, Rab GTPases cycle between the GDP- and GTP-bound states, which is controlled by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs).^6-10 In general, Rab GTPases in the GDP-bound state are extracted from membranes by GDP dissociation inhibitors (GDIs) via interacting with the hydrophobic prenyl groups of Rab GTPases. This interaction not only keeps Rab GTPases in the cytoplasm but also controls their recruitment onto the correct subcellular location.^8,11 When in the GTP-bound state, Rab GTPases can bind to diverse downstream effectors which may regulate cargo selection, vesicle formation, sorting, motility, tethering and fusion with the appropriate membranes.^12-16

Several Rab GTPases have been demonstrated to regulate intracellular trafficking of GPCRs. The most studied Rab GTPase in GPCR trafficking is Rab5, a well characterized GTPase involved in protein trafficking between the plasma membrane and endosomes. Rab5 has been shown to regulate the agonist-evoked internalization of multiple GPCRs.^17-27 Constitutive transport of some GPCRs from the cell surface to endosomes may also use the Rab5-mediated pathway.^28,29 In contrast, Rab4 and Rab11 are mainly involved in the recycling of internalized receptors from endosomes back to the cell surface membrane, either at distinct recycling steps or via different pathways.^{17-20,22,25,27,29} Rab7 has been shown to participate in the transport of internalized receptors to the lysosome for degradation²⁰ and in the transport from endosomes to the TGN.²⁸

Our laboratory has focused efforts on understanding the role of Rab GTPases in the biosynthesis pathway of GPCRs.³⁰ We and others have demonstrated that 3 Rab GTPases Rab1, Rab2 and Rab6, which have been known to be involved in vesicle-mediated transport along the early secretory pathway, are required for the ER-to-Golgi transport of GPCRs, including α₁-adrenergic receptor (AR), α₂-AR, β₁-AR, β₂-AR, angiotensin II type 1 receptor (AT1R), AT2R and human calcium-sensing receptor.^31-40 The following sections will discuss the role of Rab8 and Rab26 in GPCR transport from the Golgi to the cell surface and the possible underlying mechanisms.

Role of Rab8 and Rab26 in the post-Golgi transport of GPCRs

There are 2 Rab8 isoforms, Rab8A and Rab8B, which share 83% identity in their amino acid sequences and have undistinguishable functions. Rab8 isoforms are localized to different subcellular compartments (the Golgi apparatus, plasma membranes, and endosomes) and cell structures (filopodia, lamellipodia, protrusions, ruffles and primary cilia). Rab8 function in protein transport has been extensively investigated, particularly from the TGN to the apical/basolateral membrane under polarized conditions.^41,42 Rab26 is relatively less well characterized as compared with many other secretory GTPases. Rab26 was found to be highly expressed at the Golgi⁴³ and on the parotid acinar cell secretory granules.^44,45 Rab26 was shown to regulate the maturation of secretory granules⁴⁶ and the release of amylase.^44,45

The first indication of Rab8 function in GPCR maturation was its association with the post-Golgi membranes that transport newly synthesized rhodopsin to the rod outer segments in retinal photoreceptors.⁴⁷ A subsequent study showed that expression of dominant negative Rab8 mutants disrupted the post-Golgi transport of rhodopsin in Xenopus.⁴⁸ We and others have determined the role of Rab8 in the cell surface expression of α_2B-AR and β₂-AR, 2 prototypic GPCRs which mainly couple to Gi and Gs, respectively, in mammalian cells and primary cultures of neurons.^34,49 Attenuation of Rab8 function achieved by transient expression of its dominant negative mutants and shRNA-mediated knockdown significantly reduced the cell surface expression of α_2B-AR and β₂-AR as measured by intact cell ligand binding. Confocal imaging showed that both receptors were extensively co-localized with the TGN marker p230 in cells expressing the Rab8 mutants. These studies demonstrate that Rab8 is required for post-Golgi traffic of some GPCRs.

Similar to the effect of Rab8, expression of dominant negative Rab26 mutants and siRNA-mediated depletion of Rab26 markedly reduced the cell surface expression of α_2A-AR and α_2B-AR and caused a massive accumulation of the receptors in the Golgi apparatus.⁴³ These data suggest that Rab26 may function as a crucial regulator for the Golgi-to-cell surface movement of α₂-ARs. These data also provide the first evidence indicating that Rab26 may play an important role in the post-Golgi transport of GPCRs.

Differential interaction of Rab8 and Rab26 with GPCRs

To elucidate the molecular mechanisms underlying the function of Rab8 and Rab26 in regulating GPCR transport, we determined if they were able to physically associate with the receptors. Several other Rab GTPases, including Rab4, Rab5, Rab7 and Rab11, have been described to directly interact with GPCRs. For example, Rab4 and Rab11 interact with the C-termini of AT1R, β₂-AR and thromboxane A2 receptor to regulate their recycling from endosomes back to the plasma membrane.^21,50,51 We found that Rab8 bound to α_2B-AR and β₂-AR⁴⁹ and that Rab26 interacted with α_2B-AR⁴³ as measured in different protein-protein interaction assays, including co-immunoprecipitation, GST fusion protein pulldown and bioluminescence resonance energy transfer assays.

The most interesting point regarding the interaction of Rab8 and Rab26 with GPCRs is the identification of distinct Rab-binding domains or motifs in α_2B-AR and β₂-AR. α_2B-AR has a relatively large third intracellular loop (ICL3) and short ICL1, ICL2 and C-terminus, whereas β₂-AR contains a large C-terminus and short intracellular loops. In GST fusion protein pulldown assays, Rab8-binding domains were mapped to the C-termini of α_2B-AR and β₂-AR. Site-directed mutagenesis further identified multiple residues in the fragments VFNQ and PWTQTGW of the C-terminus which modulated α_2B-AR interaction with Rab8.⁴⁹ In contrast, the dileucine (LL) motif in the C-terminus was required for the interaction of β₂-AR, but not α_2B-AR, with Rab8. The LL motif is highly conserved in the membrane-proximal C-termini of the family A GPCRs⁵² and is required for the ER export of several GPCRs, including α_2B-AR and β₂-AR.^52-55 These data suggest that Rab8 function in the post-Golgi trafficking of different GPCRs is mediated through distinct mechanisms. These data also imply that the LL motif may regulate β₂-AR transport from both the ER and the Golgi.

In contrast to Rab8 interacting with the C-termini of α_2B-AR and β₂-AR,⁴⁹ Rab26 was shown to bind to the ICL3 of α_2B-AR.⁴³ These data suggest that one GPCR may utilize different intracellular domains to interact with distinct Rab GTPases to control their export trafficking (Fig. 1). However, if Rab8 and Rab26 could simultaneously bind to α_2B-AR and how they cooperatively regulate the post-Golgi export of α_2B-AR remain unknown. It is possible that Rab8 and Rab26 mediate α_2B-AR transport along different routes (Fig. 1A). It is also possible that Rab8 and Rab26 may regulate α_2B-AR transport at different steps from different sub-compartments in the same pathway (Fig. 1B and 1C).

Figure 1. Possible mechanisms by which Rab8 and Rab26 mediate the Golgi-to-cell surface transport of α_2B-AR. A, Rab8 and Rab26 mediate 2 separate pathways that deliver the receptor to the cell surface. Rab8, in its inactive form presumably localized in the cytoplasm, interacts with the C-terminus of α_2B-AR, whereas Rab26, in its active form tethered to the membrane, binds to the ICL3 of the receptor. B and C, Rab8 and Rab26 regulate α_2B-AR transport at different steps in the same route. PM, plasma membrane.

Another striking difference between the interactions of Rab8 and Rab26 with the receptors is their activation dependence. Both α_2B-AR and β₂-AR were found to associate with Rab8, preferentially in its inactive, GDP-bound form.⁴⁹ It is possible that both receptors may function as GEFs to promote the activation of Rab8. This possibility is supported by the fact that some cargo proteins can activate transport machinery to modulate their transport. For example, AT1R is able to interact with and activate Rab5 GTPase which regulates the internalization process of the receptor.²¹ It is also possible that the receptors may function as anchoring proteins for Rab8 localization to the TGN through providing docking sites for inactive, GDP-bound Rab8. In contrast, α_2B-AR interaction with active, GTP-bound form of Rab26 was much stronger than with inactive, GDP-bound form of Rab26.⁴³ These data suggest that α_2B-AR may be a downstream effector of Rab26 and the Rab26 interaction may enhance the recruitment of the receptors onto Rab26-coordinated transport vesicles.

As discussed above, different GPCRs interact with one Rab via different motifs and one GPCR may bind to different Rab GTPases via different domains. These data, together with other studies demonstrating that GPCRs are able to directly associate with Sec24 isoforms,⁵⁶ the components of ER-derived COPII vesicles that mediate cargo transport exclusively from the ER to the Golgi, and GGA3,⁵⁷ the component of clathrin-coated vesicles that transport cargo between endosomes and the TGN, strongly support that the cargo GPCRs may physically bind to components of the transport machinery to control their export trafficking. Differential interaction of distinct GPCRs with Rab GTPases may form a unique transport machinery for each individual receptor to export to the cell surface.

Conclusions and perspectives

It is apparent that Rab GTPases control the proper GPCR expression at the cell surface and different Rab GTPases may regulate different steps in the biosynthesis and processing of GPCRs. It is also intriguing to note that some Rab GTPases may directly associate with the receptors they regulate, suggesting their specific function in GPCR trafficking. However, the exact molecular mechanisms underlying the function of Rab GTPases in GPCR export remain largely unknown. The regulatory role of Rab GTPases in the cell surface export trafficking of the GPCR superfamily has just begun to be revealed and numerous questions remain to be addressed. As only a few Rab GTPases have been investigated in GPCR transport, future studies should search for more Rabs and define the specificity between various Rab GTPases and distinct GPCR members. In addition, the function of RabGEFs and RabGAPs in the trafficking of GPCRs has not been studied. Although Rab GTPases influence the cell surface expression of GPCRs at the steady-state, their function in receptor export kinetics as well as receptor sorting to different transport routes remains unknown.

Importantly, the dysfunction of Rab GTPases are implicated in the development of human diseases but their exact roles under these pathological conditions are elusive.⁵⁸ One possibility is that Rab dysfunction may actually cause the defective trafficking of some GPCRs, leading to the disruption of GPCR-mediated signaling pathways. It should be pointed out that in addition to Rab GTPases, receptor activity modifying proteins, ER chaperones, escort proteins and gatekeepers may facilitate GPCR maturation, promote receptor delivery to the plasma membrane and stabilize receptor conformation.^59-62 To further explore the function of Rab GTPases as well as many other regulatory proteins in the export trafficking of nascent GPCRs will not only greatly enhance our understanding of GPCR targeting to their functional destinations but also reveal novel therapeutic targets for human diseases involving abnormal trafficking and signaling of GPCRs.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by National Natural Science Foundation of China Grant 81370168 (to G. Wang) and National Institutes of Health Grant R01GM118915 (to G. Wu).