Isoform-Specific Regulation of Adenylyl Cyclase Function by Disruption of Membrane Trafficking

Abstract

Oligomerization plays an important role in endoplasmic reticulum processing and membrane insertion (and ultimately in regulation of function) of a number of transmembrane spanning proteins. Furthermore, it is known that adenylyl cyclases (ACs), critical regulators of cellular functions, associate into higher order (dimeric) forms. However, the importance of these higher order aggregates in regulating adenylyl cyclase activity or trafficking to the cell membrane is unclear. Therefore, we examined the potential role of oligomerization in the membrane trafficking of adenylyl cyclase. For this purpose, the ability of full-length adenylyl cyclase and various truncation mutants to self-assemble and to be targeted to the cell membrane was assessed. A truncation mutant comprised of the initial six transmembrane spanning domains and half of the C1 catalytic domain coimmunoprecipitated with full-length AC VI. Using both biotinylation assays and assessment of enzyme distribution using sucrose density gradients, we demonstrate that expression of this mutant in human embryonic kidney 293 cells impaired the ability of AC VI to traffic to the plasma membrane. Furthermore, mutant expression resulted in a significant reduction in adenylyl cyclase activity. The decrease in AC VI membrane expression was not caused by alterations in enzyme transcription. The effect of the mutant was specific for the AC V and VI isoforms and expression of the transmembrane M1 domain but not the C1a domain was required for the mutant to affect adenylyl cyclase activity. In aggregate, these data suggest that alterations in the ability of adenylyl cyclases to form higher order forms regulate both enzyme trafficking and enzyme activity.Adenylyl cyclases comprise a large family of enzymes that catalyze the synthesis of cyclic AMP from ATP. Adenylyl cyclases constitute the "effector" component of transmembrane signaling pathways that are both positively and negatively regulated by the activity of heterotrimeric G protein and G protein-coupled receptors (GPCRs). The activity of the enzyme adenylyl cyclase is itself regulated by both extracellular stimuli (via GPCR receptor activation) and intracellular stimuli (Cooper et al., 1995;Hanoune and Defer, 2001;Patel et al., 2001). Studies of the regulation of adenylyl cyclases initially focused "upstream" of the enzyme (i.e., on characterization of regulation of enzyme function via G protein and GPCR function/expression) or on the regulation of substrate (Mg 2ϩ /ATP) availability. More recently, the importance of regulation of enzyme activity by covalent modifications (i.e., enzyme phosphorylation) has increasingly been appreciated

Adenylyl Cyclase Isoform–Selective Regulation of Vascular Smooth Muscle Proliferation and Cytoskeletal Reorganization

“…HEK293 cells were transiently transfected with pcDNA3 containing either GFP or Flag-tagged AC1 (as representative of the AC1, AC3, AC8 subfamily), AC2 (as representative of the AC2, AC4, AC7 subfamily), and AC5 or AC6 cDNA using calcium phosphate as previously described. 9,12 Cells were harvested 48 to 72 hours after transfection for assessment of biological functions. For AC/extracellular signal-regulated kinase (ERK) interaction experiments, whole cell extracts were subjected to immunoprecipitation with either anti-Flag M2 (Sigma, St Louis, Mo) or anti-ERK antibodies.…”

“…Suppression of PDE in these experiments was achieved by including excess unlabeled cAMP in our permeabilization medium, as described previously. 9,12 Alternatively, assessment of cAMP production was determined via monitoring the conversion of [ 3 H]ATP to [ 3 H]cAMP with modifications as previously described. 14 Briefly, AC isoforminfected smooth muscle cells or HEK293 cells were incubated for 4 hours with [ 3 H]adenine (Ϸ3 Ci/mL; NEN).…”

New insights into the regulation of cAMP synthesis beyond GPCR/G protein activation: Implications in cardiovascular regulation

“…In particular, expression of an inactive truncated form of AC6 has been demonstrated to reduce AC8 function (Gu et al, 2002). Additionally, expression of a truncation mutation of AC6 impairs AC function in HEK 296 cells (Ding et al, 2005). This AC6 truncation mutant coimmunoprecipitates with wild-type (full-length) AC6 and decreases the plasma membrane expression of the enzyme and consequently enzyme activity.…”

“…For AC6, formation of higher order aggregates is dependent on the integrity of the first (Ding et al, 2005). For AC8, enzyme aggregation is mediated via the TM2 domain (Gu et al, 2001).…”

“…However, recent findings have emphasized that there are an increasing number of mechanisms that regulate cAMP synthesis beyond G protein regulation of catalytic activity, including: a) impact of specific adenylyl cyclase isoforms on different responses in a single cell (Gros et al, 2006), b) regulation of enzyme trafficking via the formation of higher order enzyme aggregates (Ding et al, 2005), and c) raf kinase-mediated regulation of AC function (Ding et al, 2004). These discoveries have underscored the importance of mechanisms of regulation of adenylyl cyclase function-beyond the kinetic changes in the enzyme affected by interactions with GPCR-regulated G proteins.…”