Soluble guanylyl cyclase (sGC) is a ubiquitous enzyme that functions as a receptor for nitric oxide. Despite the obligate heterodimeric nature of sGC, the sequence segments mediating subunit association have remained elusive. Our initial screening for relevant interaction site(s) in the most common sGC isoenzyme, α1β1, identified two regions in each subunit, i.e. the regulatory domains and the central regions, contributing to heterodimer formation. To map the relevant segments in the β 1 subunit precisely, we constructed multiple N- and C-terminal deletion variants and cotransfected them with full-length α1 in COS cells. Immunoprecipitation revealed that a sequence segment spanning positions 204-408 mediates binding of β1 to α 1. The same region of β1[204-408] was found to promote β1/β1 homodimerization. Fusion of β1[204-408] to enhanced green fluorescent protein conferred binding activity to the recipient protein. Coexpression of β 1[204-408] with α1 or β1 targeted the sGC subunits for proteasomal degradation, suggesting that β 1[204-408] forms structurally deficient complexes with α 1 and β1. Analysis of deletion constructs lacking portions of the β1 dimerization region identified two distinct segments contributing to α1 binding, i.e. an N-terminal site covering positions 204-244 and a C-terminal site at 379-408. Both sites are crucial for sGC function because deletion of either site rendered sGC dimerization-deficient and thus functionally inactive. We conclude that the dimerization region of β1 extends over 205 residues of its regulatory and central domains and that two discontinuous sites of 41 and 30 residues, respectively, facilitate binding of β1 to the α1 subunit of sGC.
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