The activity of the enzyme adenylyl cyclase is regulated by both extracellular stimuli (via GPCR receptor activation) and intracellular stimuli. However, the regulation of enzyme function via regulation of enzyme translation and enzyme trafficking to the plasma membrane has been largely unexplored. It is known that the adenylyl cyclase enzyme associates into higher order (dimeric) forms. However, the importance of these higher order aggregates in regulating enzyme activity is unclear. Further, the importance of forming symmetrical higher order aggregates (e.g, homodimers vs heterodimers), as a determinant of adenylyl cyclase trafficking to the cell membrane is unknown. Therefore, we examined the regulation of membrane expression of adenylyl cyclases utilizing truncation mutants of the enzyme. A truncation mutant of adenylyl cyclase isoform VI, comprising the initial six transmembrane spanning domains and half of the C1 catalytic domain, co-immunoprecipitated with wild type adenylyl cyclase VI. In HEK 293 cells, expression of this truncation mutant resulted in a significant reduction in adenylyl cyclase activity (either alone or with co-expression of wild type adenylyl cyclase VI). The effect of the mutant was specific for the adenylyl cyclase VI isoform and was not replicated by either smaller truncation mutants or by truncation mutations incorporating the TM2/C2 domains of the enzyme. Further, the mutant enzyme decreased the monomeric expression of wild type adenylyl cyclase VI as detected on immunoblots. This apparent decrease in expression was not related to either a reduction in enzyme transcription or enhancement of enzyme degradation. Utilizing both biotinylation assays and assessment of enzyme distribution using sucrose density gradients, we demonstrate that expression of the mutant impaired the ability of adenylyl cyclase VI to traffic to the plasma membrane. In aggregate, these data suggest that alterations/disruption of the ability of adenylyl cyclases to form higher order forms regulate both enzyme trafficking and enzyme activity.

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