Insights into signal transduction by the guanylyl cyclase C receptor : Role of ATP and tyrosine phosphorylation

Abstract

Signal Transduction Mediated by Guanylyl Cyclase C: Regulation by ATP and Tyrosine Phosphorylation

Introduction All living cells respond to extracellular signals by generating specific cellular responses through signal transduction. Cyclic GMP (cGMP) is a ubiquitous second messenger involved in diverse pathways. It is synthesized from MgGTP by guanylyl cyclases, which exist in two forms: soluble (activated by nitric oxide) and membrane-bound (serving as receptors for polypeptide ligands).

Guanylyl Cyclase C (GCC) is a membrane-bound receptor guanylyl cyclase, initially identified as the receptor for heat-stable enterotoxin (ST) peptides secreted by enterotoxigenic bacteria. GCC is expressed on the apical surface of intestinal epithelial cells and binds endogenous ligands such as guanylin, uroguanylin, and lymphoguanylin, elevating intracellular cGMP levels. This activates cyclic nucleotide-dependent protein kinases, which phosphorylate and activate the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), leading to chloride ion efflux. While ST peptides cause diarrhoea, endogenous ligands regulate salt and water homeostasis.

Despite GCC’s role in enterotoxigenic diarrhoea, GCC knockout mice appear normal, raising questions about its physiological importance. This thesis investigates the molecular mechanisms of GCC activation, focusing on ATP regulation and tyrosine phosphorylation.

Key Findings Role of ATP in GCC Regulation GCC contains a protein kinase-like domain (PKLD) between the extracellular ligand-binding domain and the cyclase domain.

Although PKLD lacks catalytic activity, it binds ATP.

ATP potentiated ST-stimulated GCC activity but inhibited ligand-independent activation by detergents or MnGTP.

Site-directed mutagenesis of Lys516 in PKLD abolished ATP modulation, confirming PKLD’s role in ATP binding and regulation.

ATP also altered GCC oligomeric status, converting higher-order oligomers to dimers, suggesting a structural mechanism of regulation.

Tyrosine Phosphorylation of GCC Sequence analysis identified consensus sites for tyrosine phosphorylation.

GCC was phosphorylated by EphB1 and Src family kinases (e.g., Hck, c-Src).

Tyr820 in GCC was confirmed as a strong substrate for Src family kinases.

Western blot analysis of T84 and HEK293 cells showed GCC tyrosine phosphorylation in vivo.

This is the first demonstration of tyrosine phosphorylation in receptor guanylyl cyclases.

Interaction with Src SH2 Domain Tyrosine-phosphorylated GCC peptides bound specifically to the SH2 domain of Src.

Full-length GCC interacted with Src in mammalian cells, confirmed by co-immunoprecipitation.

Interaction of GCC phosphopeptides with Src family kinases enhanced kinase activity, suggesting GCC phosphorylation may activate Src signaling pathways.

Conclusions The PKLD of GCC plays a critical structural role in transmitting ligand-binding signals to the cyclase domain via ATP binding.

Tyrosine phosphorylation of GCC represents a novel regulatory mechanism, enabling interactions with Src family kinases.

These findings reveal a previously unknown signaling pathway involving GCC and Src, with implications for cGMP regulation, chloride secretion, and broader cellular functions.