Adenylyl Cyclases (AC) activity exists in a broad range of procaryotic to eucaryotic species in which cyclic AMP (cAMP) controls many major cellular functions. The cAMP has been the prototypical second messenger, translating the actions of numerous extra cellular effectors into consequences for virtually every aspect of cellular function. Mammalian adenylate cyclases vary significantly outside of their largely conserved catalytic domains. This diversity underlies a broad range of individual regulation. It can be partitioned to discrete regions of the cell with numerous regulatory consequences. The pioneering work of Buxton and Brunton in cardiomyocytes was the first to provide compelling experimental evidence of the existence of compartmentalization as an essential feature in the action of cAMP, showing that adreno receptor mediated stimulation of AC activates a particulate pool of protein kinase A (PKA), whereas prostaglandin (PG) E1 stimulation activates cytosolic PKA. However, only relatively recently has it begun to be understood how a combination of protein-protein and protein-lipid interactions contributes to the manifestation of compartmentalization. From regulation by G-protein-coupled receptors (GPCRs) to activation of cAMP effectors and signal termination by phosphodiesterases (PDEs), the cAMP message is governed by sophisticated protein scaffolding systems. How GPCRs, cAMP-dependent PKA, A-kinase-anchoring proteins and PDEs are organized. In this article, the author summarize recent findings about ACs with regard to oligomeric assemblies, protein-lipid interactions and protein-protein interactions - which, together, define how ACs partake in higher-order signalling complexes and contribute to cAMP compartmentalization.

The transmembrane domains of ACs determine quaternary structure and plasma membrane targeting the dimerization or oligomerization is an essential feature in AC regulation. Intermolecular dimerization or hetero-oligomerization, which governs the formation of the AC catalytic core and regulates the trafficking of ACs to the plasma membrane. The formation of oligomers might also enable the incorporation of ACs into higher-order cAMP signalling modules. The nine membrane-bound ACs are large proteins (120-140 kDa) that share a common secondary structure comprising an intracellular N-terminus, two cassettes of six transmembrane domains in tandem separated by a cytoplasmic loop, termed the C1 domain, and a C-terminal cytoplasmic C2 domain. The ATP-binding C1a and C2a domains are the most conserved regions between AC isoforms, and their interaction forms the catalytic core. The transmembrane domains are not essential for catalytic activity because the C1a and C2a domains can be over expressed separately in vitro to form a forskolin stimulable enzyme. However, by their association, the transmembrane domains have a crucial role in increasing the relative concentrations of their attached catalytic domains so that they associate to form the catalytic core. The two cassettes of six transmembrane domains are complementary within the context of individual AC isoforms. Various green fluorescent proteins (GFP)-tagged, truncated AC8 molecule combinations. This approach showed that intramolecular interactions between the two transmembrane cassettes are crucial for the delivery of AC8 to the plasma membrane in live cells. When either of the two six-pass transmembrane cassettes was expressed alone, localization was restricted to the endoplasmic reticulum (ER). However, targeting to the plasma membrane was seen upon co-transfection of the partnering six-pass transmembrane cassette, independent of the associated cytoplasmic domains. One half comprising the N-terminus linked to the first transmembrane cassette linked to the first cytoplasmic loop, and the other half comprising the second transmembrane cassette linked to the second cytoplasmic loop; yield functional activity equivalent to that of full-length molecules.

Crucial determinant of AC regulation
The local membrane environment is a crucial determinant of AC regulation. Two general mechanisms organize proteins in signaling modules. ACs dimerize via their transmembrane-spanning domains. AC8 associates internally by interactions between the first and second transmembrane-spanning cassettes (TM1 and TM2, respectively), or externally by interactions between the divalent second transmembrane-spanning cassettes. The structure shared by all ACs comprises an intracellular N-terminus (Nt) and two cassettes of six transmembrane domains in tandem (TM1 and TM2), which are separated by the cytoplasmic C1 domain and the C-terminal cytoplasmic C2 domain. The C1a and C2a domains are the most conserved regions between AC isoforms, and their interaction forms the catalytic core.

The second is a more passive mechanism that relies on signalling proteins being in close proximity but not in a pre-existing interacting complex. This arrangement increases the concentration of the reactants within the local environment, enabling efficient information exchange but in a more fluid manner. Both systems apply to the regulation of ACs by GPCRs and Ca2+, which relies not only on the makeup of the immediate protein environment but also on the discrete membrane localization. We have learned a lot about the regulation of Ca2+-sensitive adenylate cyclases over the last few years - both at the biochemical and cell-biological levels. From a biochemical viewpoint it will be of considerable interest to know what makes some cyclases subject to high-affinity inhibition by Ca2+ and others insensitive, given that adenylate cyclases are at their most conserved in their catalytic domains. However, there are significant differences in the amino acids surrounding the catalytic aspartates between different isoforms. Ideally, crystal structure comparisons of Ca2+- sensitive and Ca2+- insensitive forms will reveal the underlying mechanisms. It will also be of considerable interest to determine the role of the N-terminal binding of calmodulin by AC8 - whether, for instance, it plays a recruiting role for calmodulin or, perhaps, induces a conformation in the N-terminus that allows interactions with scaffolding proteins. Newly emerging studies suggest that higher-order assemblies of adenylate cyclase molecules can occur. This propensity may be enlightening in terms of identifying partners in signaling complexes. The mechanism underlying the apposition of cyclases with CCE channels remains a burning question, whose solution may yet provide unsuspected insights into CCE organization. Continued application of single-cell methods to the measurement of cAMP is expected to provide novel insights into the dynamics of the second messenger near the plasma membrane. It was predicted some time ago with rather conservative modeling, and again more recently, that the interaction between Ca2+ and Ca2+-sensitive adenylate cyclases could yield cAMP oscillations. If the current technologies support these predictions, the next challenge will be to develop strategies to search for potential targets of cAMP spikes.

ACs is re-emerging as central molecules that dictate the compartmentalization of the cAMP message. A complex interplay of intramolecular and intermolecular interactions governs the regulation of catalytic activity, trafficking, membrane localization and higher-order associations. The dimerization of ACs might be the key property that enables their incorporation into larger multimeric complexes and, in combination with isoform specific regulators and plasma membrane compartmentalization; this could govern the organization of ACs in signalling systems. It remains a challenge to identify how cAMP signalling systems are pieced together precisely and, simultaneously, to devise pharmacological strategies that intervene in this organization from a therapeutic perspective.

Activation of adenylyl cyclases results in activation of intracellular accumulation of second messenger cAMP which functions almost exclusively through cAMP dependent protein kinase A. There is increasing awareness of the compartmentalization of cAMP signalling - the means by which cAMP levels change in discrete domains of the cell with discrete local consequences. Current developments in understanding the organization of AC in the plasma membrane are illuminating how the earliest part of cAMP compartmentalization could occur. Here focus is on recent findings regarding three levels of adenylyl cyclase organization - oligomerization, positioning to lipid rafts and participation in multi protein signalling complexes. This organization, coupled with the role of scaffolding proteins in arranging the downstream effectors of cAMP, helps to identify complexes that greatly facilitate the translation of enzyme activation into local consequences.

(Anantha Naik Nagappa is with Manipal College of Pharmaceutical Sciences, Manipal 576104. Priti Kaushik is with Birla Institute of Technology and Science, Pilani 333 031).