Claudette Klein
Professor of Biochemistry and Moleculary Biology

EDUCATION:
Ph.D., 1972, University of California, San Francisco

MEMBERSHIPS:
American Society for Biological Chemistry and Molecular Biology Protein Society

Publications   CV 
RESEARCH SUMMARY:
With the discovery of cAMP as a second messenger for a variety of hormones, identification and characterization of the signaling pathways leading to changes in adenylyl cyclase (AC) activity have been intensely studied for over 40 years. Protein purification and partial sequencing led to the eventual cloning of one AC isoform. Subsequently, nine isoforms of the mammalian enzyme have been cloned. Northern analyses and in situ hybridization have indicated that each isoform has a unique pattern of expression. The tissue distribution can be wide, as for the type 9 isoform, or more restricted as for the type 5 isoform which appears to be expressed only in heart and brain. Additionally, more than one isoform is often expressed in a cell. In light of their varied and complex modes of regulation, ACs have been proposed to serve as coincidence detectors. The enzymes are acted upon simultaneously by various hormones, and then submit the results of these influences to the cell via changes in cAMP. The response to two different signals could result in a synergistic response. Alternatively, discordant coincidence detection could occur by which the response to one signal is attenuated by the concurrent presence of a second signal.

In our studies of AC regulation, we observed that the addition of nitric oxide (NO) donor compounds or NO gas to the lower eukaryote D. discoideum inhibits their aggregation via cAMP pulses, and does so independently of any changes in cGMP or guanylyl cyclase (GC) activity. The sum of the data indicated that NO specifically alters either a regulatory domain of the AC itself or a distinct regulatory component. We continue to study the role of NO, both as a regulator of AC activity and the developmental program of this multicellular organism.

We have also demonstrated the addition of NO to cultures of N18TG2 neuroblastoma cells inhibits the accumulation of cAMP in response to either hormone or forskolin stimulation. Biochemical and molecular data indicate that the predominant AC isoform in N18TG2 cells is the type 6 and that the enzyme itself is the target of NO. NO has received a great deal of attention in recent years. It has been implicated as a regulator of vasodilation, synaptic plasticity, and immune defense. In each case, however, NO has potentially deleterious effects should its production be disturbed, as occurs in excitotoxicity and ischemia. We have described a novel coincidence detection system for AC: the reversible suppression by NO of the enzymeÕs responses to stimulatory input. Our studies will provide novel insights into the isoform-specific regulation of AC and the structural features of the enzyme underlying that regulation. They will also delineate the conditions necessary to observe that regulation in intact cells and thus implicate the physiological states under which AC regulation by NO could be significant. With the recognized importance of AC as a common focal point for signal transduction and cross-talk, and with the increasing awareness of the roles of NO as an extracellular mediator, these investigations will provide novel insights into the sophisticated regulation of each of these players and into the integration of their respective information. We will elucidate a previously unappreciated mechanism of AC regulation, one that could underlie the modulation of hormone responsiveness by NO in a variety of situations.