Towards a molecular understanding of gastrointestinal disorders due to mutations in receptor Guanylyl Cyclase C

Abstract

Towards a molecular understanding of gastrointestinal disorders due to mutations in receptor Guanylyl Cyclase C Receptor guanylyl cyclase C (GC-C) is a complex multidomain protein receptor characterised by its guanylyl cyclase enzymatic activity. It is primarily expressed on the apical surface of the intestinal epithelium, where it plays a crucial role in maintaining intestinal homeostasis through the regulation of essential processes, including fluid-ion balance, cellular differentiation, inflammatory responses, the intestinal microbiome, epithelial barrier function, and visceral pain modulation. Dysregulation of GC-C activity is associated with a range of gastrointestinal disorders, such as diarrhoea, meconium ileus, inflammatory bowel diseases (IBD), and various malignancies. These conditions can manifest as either familial syndromes or de novo occurrences. Current therapeutic strategies for these complex diseases are predominantly indirect, focusing on symptomatic relief through measures such as fluid and ion replenishment and invasive surgical interventions rather than explicitly targeting GC-C. To facilitate the development of GC-C-specific therapeutic approaches, it is imperative to elucidate the molecular mechanisms underlying these pathologies. Therefore, a panel of patient-derived mutations from published literature and clinical collaborations was assembled. A functional assessment of these mutations across the distinct domains of GC-C was conducted to establish causal relationships and gain insights into their pathological implications. Notably, mutations within the kinase homology domain (KHD) and linker region influenced the activity of adjacent domains. Given that the KHD serves as an allosteric regulator of GC-C and shares amino acid sequence similarity with tyrosine kinases, FDA-approved tyrosine kinase inhibitors were screened, and they were found to effectively attenuate the guanylyl cyclase activity of both wild-type and hyperactive disease variants of GC-C. These findings provide preliminary evidence suggesting that the KHD within GC-C may represent a promising target for identifying regulators of its activity with potential therapeutic applications. Additionally, murine intestinal organoids, generated from various models of differential GC-C activity, were characterised to evaluate their potential as a model system for gaining epithelial insights into GC-C dysfunction. The organoid model expressing a hyperactive variant of GC-C, which corresponds to the patient-specific variant identified in over 30 individuals with diarrhoea and inflammatory bowel disease (IBD), exhibited significantly elevated levels of cellular cyclic GMP and demonstrated heightened sensitivity to both the endogenous ligand and bacterial toxin. Furthermore, transcriptomic analysis of this hypersensitive model revealed transcriptomic profiles that closely resemble those observed in IBD patient’s ileal biopsies and mouse models exhibiting ileal dysbiosis. This study advances the molecular understanding of GC-C-related pathologies and is expected to enhance the management of analogous cases in clinical practice. Additionally, it establishes a foundation for developing personalised, targeted therapies aimed at modulating GC-C signalling in disease states associated with its dysfunctional variants.