G proteins and regulation of adenylyl cyclases

Members of two classes of G proteins, G_s and G_i regulate adenylyl cyclases directly. The activated α subunit of G_s stimulates all mammalian adenylyl cyclases, while the α subunits of the three G_i proteins inhibit certain isoforms. Other adenylyl cyclases are inhibited by G protein βγ subunits or are activated conditionally by βγ in the presence of G_sα. We are taking structural and mutagenic approaches to understand these regulatory interactions. High resolution crystal structures of G_iα1 in its GTPγS, GDP, and GDP + AlF₄- bound states have been solved, as has the structure of the G_iα1β₁γ₂ heterotrimer. The conformational changes that drive the sequential protein-protein interactions that characterize the system and that permit slow hydrolysis of GTP have been visualized. The G protein β subunit contains 7 so-called WD repeats and its structure is that of a 7-fold β propeller. Biochemical and structural approaches to adenylyl cyclases are limited by the low abundance and hydrophobicity of these enzymes. We have synthesized and purified a 55-kDa soluble form of mammalian adenylyl cyclase devoid of all of its hydrophobic domains. This protein retains the regulatory features that are shared by mammalian adenylyl cyclases. The two cytosolic domains of the enzyme can also be synthesized separately, purified, and reconstituted to yield G_sα- and forskolin-stimulated enzymatic activity. This system will greatly facilitate biochemical, genetic, and structural approaches to understanding mechanisms of regulation.