Elsevier

Cellular Signalling

Volume 21, Issue 4, April 2009, Pages 488-501
Cellular Signalling

Intracellular trafficking and assembly of specific Kir3 channel/G protein complexes

https://doi.org/10.1016/j.cellsig.2008.11.011Get rights and content

Abstract

We have previously demonstrated that Kir3.1 channels and Gβ1γ2 subunits initially interact in the endoplasmic reticulum (ER). To elucidate the role that anterograde protein trafficking pathways may play in the formation of these complexes, we used dominant negative (DN) mutants of the small G proteins Sar 1 and various compartment-specific Rabs which impede anterograde protein trafficking at different steps. Sar 1 H79G and Rab 1 S25N mutants efficiently blocked the plasma membrane trafficking of the Kir3.1/Kir3.4 complex however they did not block the Gβ1γ2/Kir3.1 interaction as measured using bioluminescence resonance energy transfer (BRET). This interaction was also insensitive to the presence of DN Rabs 2, 6, 8, and 11. These results confirm that Gβγ/Kir3 complexes form early during channel biosynthesis and trafficking. Using a combination of BRET, protein complementation assays and co-immunoprecipitation, we demonstrate that Gβ1-4 can interact with Kir3.1 in the absence of Kir3.4. Gβ5 does not directly interact with the channel but can still be co-immunoprecipated as part of a larger complex. The interaction between Gβ and Kir3.1 was selectively fostered by co-expression with different Gγ subunits. When Gγ1 or Gγ11 was co-expressed with eGFP-Gβ3 or eGFP-Gβ4, the interaction with the effector was lost. Kir3.2 was capable of interacting with Gβ1-3 and not Gβ4 or Gβ5. These interactions were again fostered by co-expression with Gγ and were also insensitive to DN Sar 1 or Rab 1. Taken together, our data show that these “precocious” channel/G protein interactions are specific and may have implications beyond their basic function in signalling events.

Introduction

The classical paradigm for G protein activation of effectors (at least in the mammalian visual system) involves free diffusion of the relevant proteins at the plasma membrane. In humans, there are at least 20 different Gα subunits, 5 Gβ subunits, 13 Gγ subunits, multiple effectors and associated proteins and several hundred 7 transmembrane domain-containing receptors (7TM-Rs). Considering the fact that some 7TM-R effectors such as ion channels can be fully activated within 1 s following addition of an agonist [1], [2], this model encounters difficulties in accounting for the specificity and the rapidity of G protein signalling in other tissues (see [3] for review). A number of studies have demonstrated the existence of stable interactions, independent of receptor activation between the receptor and G protein [4], [5], G protein and the effector [6] and even between the receptor and the effector [7], [8]. If the signalling partners interact before receptor activation, the question of where these proteins initially interact together becomes critical. Recent studies have shown that many of these proteins interact initially in the endoplasmic reticulum (ER), including receptor equivalents in receptor dimers [9], receptor and Gβγ subunits [10] and effectors such as Kir3 channels and adenylyl cyclase with nascent Gβγ [6], [11], [12]. If these complexes are pre-formed during protein biosynthesis and maturation, they would need to be trafficked inside the cells as a complex and not necessarily as individual proteins.

The Kir3 family of inwardly rectifying potassium channels was among the first effectors demonstrated to be directly modulated by Gβγ [13]. To date, little specificity has been shown for the different Gβγ subunits with respect to Kir3 channel modulation in vitro, with the exception that combinations containing Gγ1 (found in the mammalian visual system) show reduced efficacy [14]. A recent study has demonstrated both constitutive and agonist-induced interactions between Kir3 channels and various Gβγ combinations in living cells using fluorescence resonance energy transfer (FRET) and total internal reflectance fluorescence (TIRF) microscopy [15]. Direct activation of Kir3 channels by Gβγ is therefore mostly independent of the subunit composition of the heterodimer with the exception of Gβ5 and Gγ1 [14], [15]. This is also generally true for other Gβγ effectors such as adenylyl cyclase [16] although there are exceptions. For example, P-Rex1 is a GEF for Rac which is activated by combinations of Gβ1-4 but not Gβ5 and these interactions have differing affinities depending on the Gγ isoform present [17]. However, most of these studies have focused naturally on interactions important for Gβγ-dependent signalling events. Here, we focus on specificity in precocious (i.e. agonist-independent) interactions between Kir3.1 or Kir3.2 channel subunits or adenylyl cyclase II (ACII) with different Gβγ combinations. Recently, the development of bioluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementation (BiFC) approaches has allowed us to study the different types of interactions important in GPCR signalling systems in living cells [18], [19]. We are now also able to link these interactions to particular trafficking steps in the anterograde trafficking of these complexes. We show here that although the trafficking of Kir3 channels is sensitive to dominant negative Sar 1 and Rab 1 GTPases, interactions between the channel and Gβγ are not, confirming our previous observations that Kir3.1 and Kir3.2 subunits and Gβγ initially interact in the ER.

Section snippets

Constructs

Constructs encoding Kir3.1-RLuc, Kir3.2-Rluc, CD4-Rluc, eGFP-tagged Gβ1-5, AC2-Rluc, β2AR-RLuc, β2AR-GFP10, Kir3.4, YFP (1–158)-Gβ1, YFP (158–239)-Gγ1, YFP (158–239)-Gγ2, YFP (158–239)-Gγ3, YFP (158–239)-Gγ7, YFP (158–239)-Gγ11, GFP10-Gγ2, soluble βARK-CT, WT and DN Sar 1 and Rab constructs were used as previously described [6], [10], [11], [12], [20], [21]. Flag-Gβ1-5, HA-Gγ1, HA-Gγ2, HA-Gγ3, HA-Gγ7, and HA-Gγ11 were obtained from UMR cDNA Resource Center (www.cdna.org). An extracellularly

Kir3.1/3.4 trafficking to the cell surface

To be transported to the cell surface, the Kir3.1 subunit must be assembled with either Kir3.2 or Kir3.4 subunits, as it lacks an ER export signal [25]. The use of dominant negative forms of the Sar 1 and Rab GTPases is now a common way to modulate trafficking itineraries of proteins during their maturation and subsequent insertion into the plasma membrane. These small GTPases regulate multiple anterograde protein trafficking events including the budding of transport vesicles from donor

Discussion

We have demonstrated that cell surface expression of Kir3.1 channels depends on Rab 1- and Sar 1-dependent anterograde protein trafficking. We confirm that channel assembly per se was not affected as even though trafficking was blocked, Kir3.1 and Kir3.4 still become associated as demonstrated using co-immunoprecipitation (Fig. 5). We and others [10], [30], [31] have previously demonstrated that the β2AR also uses these pathways. Curiously, after exit from the ER, the receptor uses a pathway

Acknowledgements

This work was supported by grants from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Quebec to T.E.H. T.E.H. is a Chercheur National of the Fonds de la Recherche en Santé du Québec. M.R. holds a doctoral award from the Fonds de la Recherche en Santé du Québec. NR is a recipient of a McGill Faculty of Medicine Internal Studentship Award. We thank Dr. Denis J. Dupré (Dalhousie University, Halifax, Canada) for generating a number of the reagents (listed in

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