title: Expression, purification, and characterization of the intra-cellular domain of the ANP receptor.
abstract: The membrane-bound atrial natriuretic peptide receptor (GCA) catalyzes the formation of cGMP from GTP in response to natriuretic peptide hormones. Previous structural studies have focused on the extra-cellular hormone binding domain of this receptor whereas its intra-cellular domain has not yet been amenable to such studies. We report here the baculovirus expression and purification of the GCA intra-cellular domain construct GCA(ID) comprising the complete intra-cellular region which includes the kinase-homology domain, coiled-coil region, and catalytic cyclase domain. The intra-cellular domain was enzymatically characterized in terms of guanylyl cyclase activity and the effects of ATP, manganese, and Triton X-100. Our results indicate that the activity of the intra-cellular domain of the ANP receptor is about 2 fold less active compared to a truncated cyclase domain construct lacking the kinase-like domain that was also expressed and purified. In addition, unlike the full length receptor, the intra-cellular domain could not be activated by Triton X-100/Mn(2+) or its activity stimulated by ATP. These data therefore indicate that the major part of the transition from the basal state to the fully, ANP/ATP-dependent, activated state as well its stimulation/enhancement by Triton X-100/Mn(2+) requires the full length receptor. These receptor insights could aid in the development of novel therapeutics as the GCA receptor is a key drug target for cardiovascular diseases.
authors.affiliation: Department of Biochemistry/RT500, Case Western Reserve University, 10900 Euclid Av., Cleveland, OH 44106, USA.
authors.forename: Priyaranjan
authors.initials: P
authors.lastname: Pattanaik
authors.forename: Laura
authors.initials: L
authors.lastname: Fromondi
authors.forename: Kwok Peng
authors.initials: KP
authors.lastname: Ng
authors.forename: Jiangyan
authors.initials: J
authors.lastname: He
authors.forename: Focco
authors.initials: F
authors.lastname: van den Akker
mesh_terms: D017434:Protein Structure, Tertiary; D017461:Receptors, Atrial Natriuretic Factor / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D017461:Receptors, Atrial Natriuretic Factor