Persistent interactions between the two transmembrane clusters dictate the targeting and functional assembly of adenylyl cyclase.
Adenylyl cyclases possess complex structures like those of the ATP binding cassette (ABC) transporter family, which includes the cystic fibrosis transmembrane regulator, the P-glycoprotein, and ATP-sensitive K(+) channels [1-4]. These structures comprise a cytosolic N terminus followed by two tandem six-transmembrane cassettes, each associated with a highly homologous (ATP binding) cytosolic loop [5-8]. The catalytic domains, which are located in the two large cytoplasmic loops, are highly conserved and well studied. The crystal structure of these domains has even been described recently [9, 10]. However, nothing is known of the function or organization of the 12 transmembrane segments. In the present study we adopted a range of strategies including live-cell fluorescence resonance energy transfer (FRET) microscopy, coimmunoprecipitation, and functional assays of various truncated and substituted, fluorescently-tagged molecules to analyze the trafficking and activity of this molecule. When expressed as individual peptides, the two transmembrane domains - largely independently of any cytosolic region - formed a tight complex that was delivered to the plasma membrane. This cooperation between the two intact transmembrane domains was essential and sufficient to target the enzyme to the plasma membrane of the cell. The extracellular loop between the ninth and tenth transmembrane segments, which contains an N-glycosylation site, was also necessary. Furthermore, the interaction between the two transmembrane clusters played a critical role in bringing together the cytosolic catalytic domains to express functional adenylyl cyclase activity in the intact cell.
Gu
C
C
Neuroscience Program, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Sorkin
A
A
Cooper
D M
DM
D000262:Adenylyl Cyclases / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004735:Energy Transfer; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D000262:Adenylyl Cyclases