A splice variant of the guanylyl cyclase-A receptor interferes with atrial natriuretic peptide (ANP) signaling

Background Activation of the homodimeric transmembrane guanylyl cyclase-A (GC-A) receptor upon binding of its extracellular ligands, atrial (ANP) and B-type (BNP) natriuretic peptides, leads to cyclic GMP formation in many types of cells. This NP/GC-A pathway has a critical role in the endocrine regulation of arterial blood pressure and volume and in the local counter-regulation of cardiac hypertrophy and fibrosis. Alterations of this system result in arterial hypertension, hypervolemia and cardiac hypertrophy. Many studies have shown that exposure of GC-A to high concentrations of ANP/BNP or to growth hormones such as angiotensin II (Ang II) or endothelin provokes homologous versus heterologous desensitization of the receptor [1]. However, the mechanisms accounting for this loss of function of GC-A in vivo are largely unknown. In the present study we identified and characterized a novel isoform of GC-A (GC-AΔLys-Gln 330) with unique structural properties. Our data reveal that this splice variant functions as dominant negative isoform and suggest that increased alternative splicing of GC-A may contribute to homologous or heterologous desensitization of the NP/GC-A system.


Background
Activation of the homodimeric transmembrane guanylyl cyclase-A (GC-A) receptor upon binding of its extracellular ligands, atrial (ANP) and B-type (BNP) natriuretic peptides, leads to cyclic GMP formation in many types of cells. This NP/GC-A pathway has a critical role in the endocrine regulation of arterial blood pressure and volume and in the local counter-regulation of cardiac hypertrophy and fibrosis. Alterations of this system result in arterial hypertension, hypervolemia and cardiac hypertrophy. Many studies have shown that exposure of GC-A to high concentrations of ANP/BNP or to growth hormones such as angiotensin II (Ang II) or endothelin provokes homologous versus heterologous desensitization of the receptor [1]. However, the mechanisms accounting for this loss of function of GC-A in vivo are largely unknown. In the present study we identified and characterized a novel isoform of GC-A (GC-AΔ Lys 314 -Gln 330 ) with unique structural properties. Our data reveal that this splice variant functions as dominant negative isoform and suggest that increased alternative splicing of GC-A may contribute to homologous or heterologous desensitization of the NP/GC-A system.

Methods and results
Reverse transcription-PCR analyses revealed a novel isoform of GC-A mRNA which is expressed in all studied murine tissues such as heart, kidney and lung, although at a ~10-fold lower level compared to the wild-type receptor. The isoform results from the deletion of a 51-bp sequence in exon 4, generated by splicing a cryptic donor site to the normal acceptor site. This alternative splicing is predicted to delete 17 amino acids (Δ Lys 314 -Gln 330 ) in the membrane-distal part of the extracellular ligand-binding domain of GC-A.
The deletion is not located directly within the ligandbinding site of GC-A (which is within the membraneproximal part of the extracellular domain) and therefore a direct influence on ANP and/or BNP binding seemed unlikely. However, molecular modeling of the extracellular domain of the splice variant indicated that subsequent alterations in the protein structure could interfere with NP binding or signaling. Indeed, our functional studies with transiently transfected HEK-293 cells demonstrated that binding of ANP and ANP-induced cyclic GMP formation by GC-AΔ Lys 314 -Gln 330 were totally abolished. Furthermore, cotransfection studies showed that this GC-A variant forms heterodimers with the wild-type receptor and inhibits ligand-inducible cGMP generation, acting as a dominant negative isoform.
To elucidate whether increased alternative splicing of GC-A could participate in the negative regulation of the NP/ from 4th International Conference of cGMP Generators, Effectors and Therapeutic Implications Regensburg, Germany. 19-21 June 2009 GC-A system by Ang II in vivo we examined the expression and activity of GC-A in lungs from control mice, and from mice treated with Ang II at a dose of 300 ng/kg/min during 7 days. Ang II -treated mice showed a markedly enhanced pulmonary mRNA expression of spliced GC-A which was concomitant to diminished GC-A/cGMP responses to ANP.
Our ongoing in vitro studies are directed to investigate whether alternative splicing of GC-A does occur in all cells expressing the receptor, or only in specific cell types. To address this question we use real-time RT-PCR to determine the ratio of wild-type versus spliced GC-A mRNA expression in different primary cultured murine cells. First results demonstrate that the splice variant is expressed in cultured microvascular lung endothelial cells (MLEC). Treatment of MLEC with ANP (100 nM during 7 h) enhanced the mRNA expression of the spliced GC-A isoform, suggesting that alternative splicing could be involved not only in heterologous (Ang II-induced) but also in homologous (NP-induced) inactivation of GC-A.

Conclusion
We conclude that alternative splicing can regulate endogenous NP/GC-A signaling. Our observations in vivo/in vitro indicate that Angiotensin II -and ANP-induced alternative splicing of GC-A may represent a novel mechanism for reducing the sensitivity of the receptor to ANP and BNP.