Smooth muscle plays an essential role in a wide variety of physiological processes, and although the basic function of every smooth muscle is the same, to contract and relax, the mechanical properties and responsiveness to hormones, neurotransmitters and drugs varies greatly between smooth muscle types. Factors that dictate the contractile characteristics of smooth muscle include plasma membrane properties, ratio and compliment of signal transducing proteins, the composition of the contractile apparatus itself. Alteration in the normal blend of these components is thought to underlie the molecular basis of several human diseases that involve smooth muscle including hypertension, bronco spasm, sexual dysfunction, gastrointestinal disorders and glaucoma. It is our hypothesis that by studying the molecular processes by which individual smooth muscles normally respond to stimulation will lead to more selective therapies to treat these disorders. The recent completion of the human and mouse genomes in combination with advanced techniques in mass spectrometry affords new opportunities for probing signal transduction pathways in cells. In this proposal we will employ a unique combination of proteomics, muscle physiology, molecular biology, immuno-histochemistry and mouse genetics to determine the molecular mechanisms by which cGMP through the activation of cyclic GMP dependant protein kinase (PKG) regulates smooth muscle relaxation. Examination of phosphoproteomes of various smooth muscles identified a distinct subset of early protein targets for PKG. Several were identified in the mouse and human genome, including CHASM, a novel protein containing a previously unidentified motif that is highly conserved in the smoothelin family of smooth muscle specific proteins. When added to permeabilized smooth muscles, CHASM causes calcium desensitization and relaxation in a phosphorylation dependant manner. The degree of sequence divergence of the intervening non-conserved amino acids within the CHASM motif region suggests that CHASM and the smoothelins may be part of a larger family of smooth muscle specific proteins that are important in the mediating the actions of cGMP/PKG. To directly test this hypothesis we have deleted the CHASM gene and obtained CHASM null mice.
