Pressure-induced stretch in the heart, both in vivo and in vitro, has been shown to stimulate anabolic processes including increased rates of ribosome and protein synthesis. In perfused rat hearts, stretch-induced anabolic responses occur concurrent with and are dependent upon accumulation of the intracellular second messenger adenosine 3', 5'- cyclic monophosphate (cAMP) and activation of cAMP-dependent protein kinase. It has been the continuing goal of this project to identify the molecular requirements for accumulation of the intracellular second messenger cAMP in response to cell deformation. Prior to the results from work funded by this grant, no information was available regarding the isolation and purification of the specific mechanosensitive element of any mechanoresponsive signal transduction pathway. Work in a simple cell model, hypotonic swelling of S49 mouse lymphoma cells, has delineated the components of a signal transduction pathway responsible for cAMP accumulation following cell deformation. Mechanical forces stimulate cAMP accumulation through direct acceleration of adenylyl cyclase activity. This stimulation appears to occur independent of the action of the guanine nucleotide-binding regulatory proteins involved in hormonal regulation of adenylyl cyclase activity. Currently no information is available as to the structure/function relationships which impart the capacity to transduce deformation stimuli into the generation of an intracellular signal. It is the goal of the work proposed in this continuation application to obtain structure/function information for adenylyl cyclase. Recent work has shown that adenylyl cyclase exists in multiple isoforms in mammalian cells, with no specific isoform thus far being exclusively found in mechanoresponsive tissues or cells. Initial work on the continuation of this project has involved identifying, cloning, and sequencing of isoforms from mechanoresponsive cells. Once these and other isoforms are cloned, they are expressed in cells which lack mechanoresponsive cAMP accumulation and tested to determine the conservation of the mechanoresponsive phenotype in the adenylyl cyclase family. Thus far, Type I and Type III isoforms have been tested, with only Type I demonstrating mechanoresponsive behavior. Once isoforms are sequenced and tested for mechanoresponsiveness, conserved and divergent structures will be identified and domains selected which correlate to this property. Genetic manipulation of these conserved domains and subsequent expression of mutated adenylyl cyclases in the cell systems described above will be used to test the participation of these domains in mechanoresponsive behavior.
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