Isoform-specific regulation of adenylyl cyclase function by disruption of membrane trafficking.
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.
Ding
Qingming
Q
Robarts Research Institute, 100 Perth Dr., London, ON, Canada N6A 5K8.
Gros
Robert
R
Chorazyczewski
Jozef
J
Ferguson
Stephen S G
SS
Feldman
Ross D
RD
D000262:Adenylyl Cyclases / Q000378:metabolism* / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D014162:Transfection / Q000379:methods
D007527:Isoenzymes; D000262:Adenylyl Cyclases