Allostery, Protein Complexes and Cellular Distribution for the Nitric Oxide Receptor, Soluble Guanylyl Cyclase

Project Co-Leaders: Matthew J. Gage, NAU and William R. Montfort, U of A

ABSTRACT:

Nitric oxide (NO) is a small, reactive molecule involved in numerous signaling pathways, including those regulating angiogenesis and metastasis. Vascular endothelial growth factor (VEGF), which is integral to tumor vascularization, stimulates cell migration using NO signaling. The primary cellular receptor for NO is soluble guanylyl cyclase (sGC), a heterodimeric hemoprotein of 150 kDa and an attractive target for the treatment of disease, including cancer. Binding of NO stimulates sGC activity, leading to the establishment of a cGMP signaling cascade. sGC is allosterically regulated by a variety of molecules, including NO, ATP, YC- 1 (a small nucleotide-like pharmacophore), and by posttranslational modifications. Despite extensive study, little is known about the overall shape of sGC, the means by which allosteric regulation takes place, the arrangement of functional domains in the protein or the arrangement of the protein in the cell. We intend to fill this gap through fluorescence-based approaches that will allow us to measure structural changes within sGC, and also to monitor sGC activity and localization within the cell. Specifically, we intend to incorporate paired fluorophores to full-length and truncated forms of sGC such that FRET measurements will reveal the distances between functional domains under stimulating and inhibiting conditions. We have developed a robust model system involving sGC from the hawkmoth (Manduca sexta) with which to begin the design studies; intracellular studies will be performed with human sGC. Although funding has only been in place for 3 months, we have made several discoveries since submitting our original proposal that provide the foundation for our second year of pilot project funding. First, we have developed robust expression of the two ~20 kDa PAS domains of sGC and, importantly, discovered that molecule YC-1 binds to the alpha subunit PAS domain. We will focus on PAS domain structure, folding, YC-1 binding, domain-domain interactions and location in the larger protein. Second, we have obtained a molecular envelope for a 92 kDa sGC fragment that contains the NO and YC-1 binding sites, using smallangle X-ray scattering (SAXS). We will localize each domain in this envelope to develop the first molecular model of sGC. Third, we have discovered that regulation of cytosolic sGC activity by extracellular thrombospondin 1 occurs via calcium influx, thus linking two important signaling pathways. We will uncover the molecular mechanism behind this new discovery. And fourth, we have demonstrated that sGC displays a punctate arrangement in both primary smooth muscle cells and immortalized cancer cells in tissue culture, but the functional consequences of this stark pattern are unknown. Tagging sGC with a GFP variant allows us to follow sGC movement in live cells. We will uncover the means of cellular localization and how it changes in response to extracellular stimuli. The results generated from this pilot project will provide the basis for future collaborative funding and research efforts.

Specific Aims:

1. To determine the domain organization and structural transitions in activated and inhibited sGC
2. To determine the nature of structural and spatial transitions in live cells under activating and inhibiting conditions