Auto-inhibition of adenylyl cyclase 9 (AC9) by an isoform-specific motif in the carboxyl-terminal region

Abstract

Trans-membrane adenylyl cyclase (tmAC) isoforms show markedly distinct regulatory properties that have not been fully explored. AC9 is highly expressed in vital organs such as the heart and the brain. Here, we report that the isoform-specific carboxyl-terminal domain (C2b) of AC9 inhibits the activation of the enzyme by Gs-coupled receptors (GsCR). In human embryonic kidney cells (HEK293) stably overexpressing AC9, cAMP production by AC9 induced upon the activation of endogenous β-adrenergic and prostanoid GsCRs was barely discernible. Cells expressing AC9 lacking the C2b domain showed a markedly enhanced cAMP response to GsCR. Subsequent studies of the response of AC9 mutants to the activation of GsCR revealed that residues 1268-1276 in the C2b domain were critical for auto-inhibition. Two main species of AC9 of 130 K and ≥ 170 K apparent molecular weight were observed on immunoblots of rodent and human myocardial membranes with NH-terminally directed anti-AC9 antibodies. The lower molecular weight AC9 band did not react with antibodies directed against the C2b domain. It was the predominant species of AC9 in rodent heart tissue and some of the human samples. There is a single gene for AC9 in vertebrates, moreover, amino acids 957-1353 of the COOH-terminus are encoded by a single exon with no apparent signs of mRNA splicing or editing making it highly unlikely that COOH-terminally truncated AC9 could arise through the processing or editing of mRNA. Thus, deductive reasoning leads to the suggestion that proteolytic cleavage of the C2b auto-inhibitory domain may govern the activation of AC9 by GsCR.

Direct stimulation of adenylyl cyclase 9 by the fungicide imidazole miconazole

“…HEK293 cells stably overexpressing mouse (Antoni et al, 1998a) or human AC 9 (Paterson et al, 2000), human AC9 truncated at start of the C2b domain (AC9_Y1242, (Pálvölgyi et al, 2018)) rat AC2 (Feinstein et al, 1991), or bovine AC1 (Krupinski et al, 1989) were produced and propagated as described previously (Antoni et al, 1998a). The cells were used between 5 to 25 passages.…”

“…cAMP accumulation in intact cells. HEK293 cells that stably overexpress tmACs at levels ~20-fold above normal cellular concentration were maintained as previously described (Antoni et al, 1998a, Pálvölgyi et al, 2018. For the measurement of cAMP accumulation the cells were plated in growth medium in 24-well tissue culture trays.…”

“…We have previously shown that the C2b domain exerts a marked auto-inhibitory effect on the activation of AC9 by Gs-coupled receptors (Pálvölgyi et al, 2018), and therefore examined the possibility that miconazole stimulated AC9 by relieving this auto-inhibition. The effects of miconazole on full-length AC9 and AC9-Y1242, a COOH-terminally truncated human AC9 that lacks the C2b domain (Pálvölgyi et al, 2018), were identical. Thus, miconazole also activates AC9 in the absence of the auto-inhibitory domain (Fig 7A).…”

“…As HEK293 cells express endogenous β2-adrenergic receptors (Rosethorne et al, 2010), the interaction between the beta agonist compound isoproterenol and miconazole was analysed. This was restricted to AC9_Y1242, because full-length AC9 is practically unresponsive to Gs-coupled receptors in HEK 293 cells (Pálvölgyi et al, 2018). As shown in Fig 7B, the effects of miconazole and a maximally effective concentration of isoproterenol (300 nM) on the production of cAMP in cells expressing AC9_Y1242 were additive (Two-way ANOVA, Interaction : F (3, 24) 0.31, P=0.82, Miconazole concentration effect : F (3,24) 206, P<0.0001, Isoproterenol effect : F (1,24) 170, P<0.001).…”

“…B) Effect of miconazole on cAMP production by HEK 293 cells expressing AC9_Y1242 in the presence of vehicle (triangles ) or 300 nM isoproterenol (squares). AC9 was not tested as it barely responds to isoproterenol (Pálvölgyi et al, 2018). Two-way ANOVA gave no interaction, the effects of miconazole (F (3, 24) = 205.6, P < 0.0001 ) and isoproterenol (F (1, 24) = 170.0, P < 0.0001) were statistically significant.…”