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A-kinase anchoring proteins (AKAPs) influence the spatial and temporal regulation of cAMP signaling events. Anchoring of PKA in proximity to certain adenylyl cyclase (AC) isoforms is thought to enhance the phosphorylation dependent termination of cAMP synthesis. Using a combination of immunoprecipitation and enzymological approaches, we show that the plasma membrane targeted anchoring protein AKAP9/Yotiao displays unique specificity for interaction and the regulation of a variety of AC isoforms. Yotiao inhibits AC 2 and 3, but has no effect on AC 1 or 9, serving purely as a scaffold for these latter isoforms. Thus, Yotiao represents an inhibitor of AC2. The N terminus of AC2 (AC2-NT), which binds directly to amino acids 808-957 of Yotiao, mediates this interaction. Additionally, AC2-NT and Yotiao (808-957) are able to effectively inhibit the association of AC2 with Yotiao and, thus, reverse the inhibition of AC2 by Yotiao in membranes. Finally, disruption of Yotiao-AC interactions gives rise to a 40% increase in brain AC activity, indicating that this anchoring protein functions to directly regulate cAMP production in the brain. cyclic AMP | protein kinase A | AKAP9

Author(s): Srikumar Sinnarajah [1]; Carmen W. Dessauer [2]; Deepa Srikumar [1]; Jun Chen [2]; John Yuen [1]; Solomon Yilma [3]; John C. Dennis [3]; Edward E. Morrison [3]; Vitaly Vodyanoy [...]

Research has been conducted on the P-site inhibitors, adenosine and adenine nucleotide analogues inhibiting adenylyl cyclase. The crystal structures of the adenylyl cyclase's catalytic core complexed with P-site inhibitors have been examined.

Recent kinetic, binding and crystallographic studies using P-site inhibitors of mammalian adenylate cyclases provide new insights into the catalytic mechanism of these highly regulated enzymes. Here, Carmen Dessauer and colleagues discuss the conformational states of adenylate cyclase, the structural determinants of inhibitor binding and the potential uses of these inhibitors as pharmacological agents.

Adenylyl cyclase activity can be reconstituted by simple mixture of the two cytosolic domains of the enzyme after their independent synthesis in Escherichia coli. We have synthesized and purified the [C.sub.1a] domain of type I adenylyl cyclase and the [C.sub.2] domain of the type II enzyme to assess their interactions with each other and with the activators [G.sub.s[Alpha]] and forskolin. In the absence of an activator, the fragments associate with low affinity and display low catalytic activity. This basal activity can be stimulated more than 100-fold by either forskolin or activated [G.sub.s[Alpha]]. Further, the addition of these activators increases the apparent affinity of the fragments for each other. Stimulation of catalysis by [G.sub.s[Alpha]] and forskolin is synergistic. These data suggest a model wherein either [G.sub.s[Alpha]] or forskolin enhances association of the other activator with adenylyl cyclase, as well as facilitating the interaction between the [C.sub.1] and [C.sub.2] domains of the enzyme.

The G-alpha subunits of heterotrimeric G proteins suppress the activity of K+ channels of the GIRK family. Free GTP-gamma-S-activated G(sub alpha-i1) subunit suppress the GIRK activity induced by G(sub beta-1-gamma-2) in the membranes of Xenopus oocytes expressing GIRK1. G(sub alpha-i2) and G(sub alpha-i3) have no effect on GIRK in Xenopus. G(sub alpha-s)-GTP-gamma-S inhibits GIRK activity partially. The coupling of G proteins to GIRKs appears to be regulated by antagonistic interactions between G(sub alpha) and G(sub beta-gamma) proteins.