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NO-sensitive guanylyl cyclase: Identification and purification of the dimerization domain

BMC Pharmacology20055 (Suppl 1) :P57

https://doi.org/10.1186/1471-2210-5-S1-P57

  • Published:

Keywords

  • Nitric Oxide
  • Guanylyl Cyclase
  • Heme Group
  • Central Amino Acid
  • Dimerization Domain

The nitric oxide (NO)-sensitive guanylyl cyclase plays a key role in diverse signalling pathways by catalysing the biosynthesis of the messenger molecule cGMP. To date, two isoforms of the enzyme (α1β1 and α2β1) are known, both of which contain a prosthetic heme group bound to the histidine 105 of the β1 subunit. The α1β1 isoform is ubiquitously expressed and is considered to be soluble, whereas the α2β1 isoform is mainly expressed in brain and is located to the membrane via interaction with PSD-95. The three-dimensional structure of NO-sensitive guanylyl cyclase has not been solved. Yet, by sequence comparison the subunits are generally divided into three domains. An N-terminal regulatory domain, a central domain postulated to be involved in dimerization and a C-terminal catalytic domain. For catalytic activity dimerization of the α and β subunit is mandatory, but until recently the regions involved in the interaction were unknown.

By coprecipitation of several deletion mutants of the α1 and β1 subunit, expressed in Sf9-cells, we have mapped the α1 dimerization domain to amino acids 61-462. Within this region, we identified two binding sites. One, as postulated, covering the central amino acids 367-462, the other, quite unexpectedly, located in the N terminus of the α1-subunit (amino acids 61-128). By itself neither region was sufficient to mediate full dimerization. Of the β1 subunit the N-terminal and central amino acids 1-385 exhibited wt-like binding to the identified α1 domain. To further characterize the short, but stable, dimeric NO-sensitive guanylyl cyclase we purified the mutant from Sf9-cells and analysed heme-coordination and binding of NO in spectrophotometric measurements. The dimeric state of the mutant was confirmed by gelfiltration analysis.

As β1 is the binding partner for both the α1 and α2 subunit, we reasoned that dimerization of the α2β1 isoform must be mediated by parts of the α2 subunit homologous to the α1 dimerization domain. Accordingly, we cloned the respective sequences of α2 (amino acids 76-501) and analysed binding of the mutant to the N-terminal and central regions of β1.

Authors’ Affiliations

(1)
Institut für Pharmakologie und Toxikologie, Medizinische Fakultät, MA N1, Ruhr-Universität Bochum, 44780 Bochum, Germany

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