Received for publication September 1, 2004.
Revised November 4, 2004.
Accepted for publication November 16, 2004.

Isoform specific regulation of adenylyl cyclase function by disruption of membrane trafficking

Abstract

Oligomerization plays an important role in ER processing and membrane insertion (and ultimately in regulation of function) of a number of transmembrane spanning proteins. Further, it is known that adenylyl cyclases (AC), 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, co-immunoprecipitated with full-length AC VI. Utilizing both biotinylation assays and assessment of enzyme distribution using sucrose density gradients, we demonstrate that expression of this mutant in HEK293 cells impaired the ability of AC VI to traffic to the plasma membrane. Further, mutant expression resulted in a significant reduction in adenylyl cyclase activity. The decrease in AC VI membrane expression was not due to alterations in enzyme transcription. The effect of the mutant was specific for the AC V and VI isoforms and expression of the TM1 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.

Key words: Adenylyl cyclases, cAMP, Receptor synthesis/trafficking