Abstract
The fidelity of cAMP in controlling numerous cellular functions rests crucially on the precise organization of cAMP microdomains that are sustained by the scaffolding properties of adenylyl cyclase. Earlier studies suggested that AC8 enriches in lipid rafts where it interacts with cytoskeletal elements. However, these are not stable structures and little is known about the dynamics of AC8 secretion and its interactions. The present study addresses the role of the cytoskeleton in maintaining the AC8 microenvironment, particularly in the context of the trafficking route of AC8 and its interaction with caveolin1. Here, biochemical and live-cell imaging approaches expose a complex, dynamic interaction between AC8 and caveolin1 that affects AC8 processing, targeting and responsiveness in plasma membrane lipid rafts. Site-directed mutagenesis and pharmacological approaches reveal that AC8 is processed with complex N-glycans and associates with lipid rafts en route to the plasma membrane. A dynamic picture emerges of the trafficking and interactions of AC8 while travelling to the plasma membrane, which are key to the organization of the AC8 microdomain.
Highlights
The residence of adenylyl cyclases (ACs) at the plasma membrane is a central premise of cAMP signalling, their presence there is much understudied
The endogenous caveolin1 stained with AlexaFluor555-conjugated goat anti-rabbit antibody, the actin filaments stained with AlexaFluor568-conjugated phalloidin and the structures stained with AlexaFluor555-conjugated wheat germ agglutinin (WGA) were visualized using the HeNe543 laser with an emission bandwidth between 573 and 630 nm
Elements of store-operated Ca2+-entry (SOCE), the AC must encounter these regulators at the plasma membrane
Summary
The residence of adenylyl cyclases (ACs) at the plasma membrane is a central premise of cAMP signalling, their presence there is much understudied. The strict dependence of AC8 on regulation by SOCE relies on direct binding of its N-terminus (AC8-Nt) to the N-terminus of Orai, which resides, along with AC8, in the so-called lipid raft domains of the plasma membrane (Willoughby et al 2012). In their natural state, biological membranes are dynamically heterogeneous, both in terms of their chemical composition and physical properties. High-resolution microscopy, mutagenesis and pharmacological tools, we suggest that AC8 assembly into lipid raft microdomains is a multi-step process that relies on numerous structural and functional elements, whose complexity has not hitherto been appreciated
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