Signalling complexes associated with adenylyl cyclase II are assembled during their biosynthesis
Introduction
Adenylyl cyclases (AC) represent a large family of enzymes that catalyze the synthesis of cyclic AMP from ATP. There are nine membrane-bound isoforms and one soluble form of mammalian AC characterized [1], [2], [3], [4], [5], [6]. AC isoforms constitute a critical effector component of transmembrane signalling pathways that are both positively and negatively regulated by the activity of heterotrimeric G proteins. Both Gα and Gβγ subunits can transduce 7-transmembrane domain-bearing receptors (7TM-Rs) signals [7], [8], [9], [10], [11]. Our understanding of AC function comes primarily from the characterization of regulatory input by G proteins and 7TM-Rs or via the regulation of substrate (Mg2+/ATP) availability. However, little is known about the assembly of nascent AC molecules with receptor and G protein or whether this occurs before their trafficking to the plasma membrane. There is increasing evidence that AC dimers may represent the basic structural unit for a functional enzyme and further that dimerization may be critical for correct plasma membrane targetting (see [12] for review). We have recently demonstrated that AC can interact directly with G protein heterotrimers, RGS proteins and the β2AR in stable, pre-associated complexes [13], [14], [15]. We have also recently demonstrated that another effector for many 7TM-Rs, Kir3 inwardly rectifying potassium channels, also interact stably with both 7TM-Rs and G proteins and that these interactions occur before trafficking of the channel to the cell surface [16]. Interestingly, the interaction between G proteins and Kir3.1 subunits is sensitive to agonist stimulation [13] even though Kir3.1 is retained intracellularly [17]. These former studies were possible because Kir3.1 subunits require additional targetting signals to reach the plasma membrane. As AC isoforms can target independently to the plasma membrane, it has been difficult to make similar assertions about where the initial interactions occur between AC and other 7TM-R signalling partners. One recent hint has come from our observation that βARs and their associated signalling machinery are found on the nuclear membranes in adult rat and mouse ventricular cardiomyocytes [18]. Stimulation of these receptors in purified nuclei leads to activation of AC suggesting that the βAR signalling complex containing AC is functional in internal compartments. However, we could not rule out that these complexes are trafficked to the nuclear membrane from the cell surface. Recent studies have demonstrated that the trafficking itinerary of angiotensin II AT1 receptors and β2AR from the ER to the Golgi is regulated in a Rab1-dependent fashion [19], [20]. We have recently used dominant-negative (DN) Rab1 and Sar1 constructs to demonstrate that the β2AR interacts with most of its core signalling partners (i.e. monomer equivalents in a receptor homodimer and Gβγ subunits but not Gα subunits) before entering the Golgi apparatus (Dupré et al., in revision, J. Biol. Chem.). In this study, we again use this approach to identify where ACII first interacts with its signalling partners. Using BRET and other biochemical techniques, we demonstrate that interactions between ACII and the β2AR or G protein occur prior to their appearance at the plasma membrane.
Section snippets
Materials and methods
Reagents were obtained from the following sources: DMEM high glucose and Lipofectamine 2000 transfection reagent were from Invitrogen Canada Inc. (Burlington, ON, Canada); fetal bovine serum and protein A-Sepharose were from Sigma-Aldrich (Oakville, ON, Canada). Unless otherwise specified, all materials were of reagent grade and were obtained from Sigma.
AC interacts constitutively with its signalling partners
We have been using BRET to study the interactions of 7TM-Rs with their various signalling partners. We now applied the same approach to study interactions with ACII. As can be seen in Fig. 1, ACII interacts constitutively (i.e. in the absence of receptor stimulation) with the β2AR (column 6) as we have shown before [15] and Gβγ subunits (columns 8 and 9, [13]). However, the interactions of ACII with either Gαs and Gαi were almost negligible (columns 13 and 14) as compared with the negative
Discussion
We have shown using a combination of biophysical and biochemical techniques that ACII is assembled with its signalling partners, receptors and heterotrimeric G proteins before it is trafficked to the cell surface. These interactions are constitutive and do not require stimulation by receptor agonist. It has become clear that 7TM-Rs are constitutively associated with effectors as parts of extensive signalling complexes [15], [44], [45], [46]. Further, there is accumulating evidence that G
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. holds a senior scholarship from the Fonds de Recherche en Santé du Québec. D.J.D. holds a postdoctoral fellowship from the Heart and Stroke Foundation of Canada. The vsvg-Sar1 WT, vsvg-Sar1 H79G and vsvg-Sar1 T39N were obtained from Dr. Phil Wedegaertner (Thomas Jefferson University, Philadelphia, PA); Flag-Rab1 N124I from Dr. Guangyu Wu (Louisiana
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These authors contributed equally to this study.