Adenylyl cyclase AC8 directly controls its micro-environment by recruiting the actin cytoskeleton in a cholesterol-rich milieu

Abstract

SummaryThe central and pervasive influence of cAMP on cellular functions underscores the value of stringent control of the organization of adenylyl cyclases (ACs) in the plasma membrane. Biochemical data suggest that ACs reside in membrane rafts and could compartmentalize intermediary scaffolding proteins and associated regulatory elements. However, little is known about the organization or regulation of the dynamic behaviour of ACs in a cellular context. The present study examines these issues, using confocal image analysis of various AC8 constructs, combined with fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. These studies reveal that AC8, through its N-terminus, enhances the cortical actin signal at the plasma membrane; an interaction that was confirmed by GST pull-down and immunoprecipitation experiments. AC8 also associates dynamically with lipid rafts; the direct association of AC8 with sterols was confirmed in Förster resonance energy transfer experiments. Disruption of the actin cytoskeleton and lipid rafts indicates that AC8 tracks along the cytoskeleton in a cholesterol-enriched domain, and the cAMP that it produces contributes to sculpting the actin cytoskeleton. Thus, an adenylyl cyclase is shown not just to act as a scaffold, but also to actively orchestrate its own micro-environment, by associating with the cytoskeleton and controlling the association by producing cAMP, to yield a highly organized signalling hub.

Store-operated Ca2+-entry and adenylyl cyclase

“…Some ACs (1/3/5/6/8) are consistently associated with rafts and others (2/4/7/9) are not [37][38][39]. A range of evidence, including live cell methods supports this assignation [39][40][41]. By traditional biochemical raft fractionation both AC8, Orai1 and TrpC1 occur in lipid rafts [27,42,43] as do CNG channels [44] and L-type channels [45].…”

“…Cooper / Cell Calcium xxx (2015) xxx-xxx [28]. The interaction can withstand Ca 2+ depletion but is disrupted by disturbing the integrity of lipid rafts or the cytoskeleton [27,28,41] (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article).…”

Interaction of membrane/lipid rafts with the cytoskeleton: Impact on signaling and function

“…The cAMP effector Epac mediates cAMP-promoted axonal attraction, while cAMP acting via PKA induces axonal repulsion[162]. Although more work is needed to identify if distinct cyclic nucleotide `pools' promote neurite growth and axonal/dendritic guidance that orignate from neuronal MLR, AC8, a neuron-specific isoform, localizes to MLR, and can tether the actin cytoskeleton to rafts[163]. Evidence does not yet exist for Cav-1/AC 8 interactions in neuronal MLR but Cav-1 and membrane cholesterol can regulate a variety of neuronal receptors and channels (e.g.,VGCC that promote cAMP production and Ca 2+ signaling[76, 164]).…”

Yeast two‐hybrid screen reveals novel protein interactions of the cytoplasmic tail of lipophorin receptor in silkworm brain

“…Thus, our yeast two-hybrid assay showed a good number of LpR4 cytoplasmic tail interacting proteins with known cell signalling functions. Adenylate cyclase is reported not just as a scaffolding protein but also actively associates with cytoskeleton to yield highly organized signalling events in cholesterol-rich milieu (Ayling et al, 2012). The cytoplasmic actin acts in cellular signal transduction pathways that involve tyrosine kinases and has a role in the organization of the cytoskeleton.…”