cGMP interacts with tropomyosin and downregulates actin-tropomyosin-myosin complex interaction.

BACKGROUND: The nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling pathway, plays a critical role in the pathogenesis of pulmonary arterial hypertension (PAH); however, its exact molecular mechanism remains undefined.

METHODS: Biotin-cGMP pull-down assay was performed to search for proteins regulated by cGMP. The interaction between cGMP and tropomyosin was analyzed with antibody dependent pull-down in vivo. Tropomyosin fragments were constructed to explore the tropomyosin-cGMP binding sites. The expression level and subcellular localization of tropomyosin were detected with Real-time PCR, Western blot and immunofluorescence assay after the 8-Br-cGMP treatment. Finally, isothermal titration calorimetry (ITC) was utilized to detect the binding affinity of actin-tropomyosin-myosin in the existence of cGMP-tropomyosin interaction.

RESULTS: cGMP interacted with tropomyosin. Isoform 4 of TPM1 gene was identified as the only isoform expressed in the human pulmonary artery smooth muscle cells (HPASMCs). The region of 68-208aa of tropomyosin was necessary for the interaction between tropomyosin and cGMP. The expression level and subcellular localization of tropomyosin showed no change after the stimulation of NO-sGC-cGMP pathway. However, cGMP-tropomyosin interaction decreased the affinity of tropomyosin to actin.

CONCLUSIONS: We elucidate the downstream signal pathway of NO-sGC-cGMP. This work will contribute to the detection of innovative targeted agents and provide novel insights into the development of new therapies for PAH.