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Our research focuses on the regulation of adenylyl cyclases, with specific emphasis on
its intracellular regulation by adenosine 3'-phosphates. Adenylyl cyclases constitute a family of
enzymes that catalyze the formation of adenosine 3':5'-monophosphate (cAMP) from 5'ATP and
are central to a major transmembrane signal transduction system. Mammalian forms of the
enzyme mediate the action of numerous stimulatory and inhibitory neurotransmitters and
hormones and acting extracellularly via cell-surface receptors that modulate guanine nucleotide-dependent regulatory (G-) proteins and thereby intracellular events. The enzyme is also
regulated directly; some isozymes are stimulated by Ca2+/calmodulin and most by forskolin.
Most are also directly inhibited by adenosine 3'-phosphates via a cytoplasmic domain (purine, or
P-site). These attenuate hormonal activation of adenylyl cyclases and may be viewed as a
counter-regulatory mechanism by which cells control susceptibility to extracellular activation by
G-protein-coupled receptors. Adenylyl cyclase isozymes exhibit different sensitivities to adenine
nucleotide 3'-phosphates that may reflect the differing physiological exigencies of the tissues
expressing the respective forms of the enzyme.
The general approach has been to use site-specific biochemical probes to define
structural characteristics of selected wild type and mutated isozymes and enzyme domains.
Studies utilize standard techniques for chemical and proteolytic fragmentation and sequence
analysis as well as time-of-flight matrix-assisted laser desorption ionization mass spectrometry,
x-ray crystallography, and biophysical measurements utilizing fluorescence. This bioorganic
approach led to the original development in our lab of two families of potent and
configuration-specific inhibitors:
b-L-adenine nucleoside 5'-polyphosphates inhibit the pre-transition form of
the enzyme and adenine nucleoside 3'-polyphosphates inhibit the post-transition form. Labeled
(32P, fluorescent) ligands have been synthesized and are used for the development of
protected 3'-nucleotide pro-drugs, configuration-selective binding assays for adenylyl cyclases,
affinity chromatography matrices, isozyme-selective ligands, and as antigenic conjugates for the
production of ligand-specific antibodies.
Future research directions center on: i) the role(s) that other domains within adenylyl cyclases play in the regulation, distribution, and expression of the enzyme, ii) the identification of other proteins and/or enzymes that interact with adenosine 3'-polyphosphates, and iii) the role(s) of adenosine 3'-polyphosphate in coordinating inhibition of adenylyl cyclase with altered cell function. These latter two areas are interdependent and are based on key structural features of adenylyl cyclases that are shared with enzymes involved in nucleic acid metabolism, e.g. the palm-domain topology common to DNA-polymerase and viral reverse transcriptase. These investigations involve collaboration with other investigators and with companies in the biotech industry. We are exploring the idea that nucleoside 3'-polyphosphates, which are naturally occurring and in some organisms are linked to developmental changes, may be the endogenous regulators whose actions are mimicked by potent anti-viral and anti-tumor agents which share structural characteristics with these nucleotides and which act on this class of enzymes. To establish this link between cellular levels of these 3'-nucleotides, adenylyl cyclase activity, and nucleic acid metabolism, and their relationship to altered patterns of cell growth and differentiation, will be to define a new intracellular regulatory pathway.
Current Abstract for NIH Grant DK38828