Molecular interaction studies of mouse secretin and angiotensin II receptors and their potential implications in water homeostasis

Abstract

Osmoregulation is critical to life and is tightly regulated by integrated physiological and behavioral responses to maintain the osmolality of body fluid. In particular, this involves recovery from dehydration both at the intracellular and extracellular levels. To achieve appropriate body fluid balance, three major hormones namely secretin (SCT), angiotensin II (ANGII) and vasopressin (VP) are responsible. Of note, SCT and ANGII share overlapping physiological roles including similar expression pattern within the brain, dipsogenic actions and activation of VP expression and/or release in mice. However, it remains unclear how their receptor pathways may cross-interact to aid osmoregulation. In recent years, G protein-coupled receptor (GPCR) oligomerization has been implicated to play roles in regulating processes such as expression, pharmacological diversity, signal transduction and internalization. Though not as extensively studied, class B GPCRs are also gaining merit in their oligomerization abilities, within which the wealth of available information is focused on SCT receptor (SCTR) homomers and heteromers. Moreover, there is also evidence indicating the ability for ANGII receptors to oligomerize. On the basis of this information, this project predominantly aims to explore the molecular association between SCTR and ANGII receptors via in vitro experiments and provide insights into its physiological relevance. In this study, bioluminescence resonance energy transfer (BRET) assays revealed SCTR and ANGII type 1a receptor (AT1aR) to form hetero-complexes. This oligomerization event was found by BRET competition to be contributed predominantly by transmembrane (TM) domain regions 2 and 4 in SCTR, and TM1 and TM4 in AT1aR. Within which, combinational use of mutant TM peptides and SCTR chimeras revealed the importance of lipid-exposed residues, particularly Leu204 and Ser205 in SCTR TM2 as key contact points for formation of the SCTR/AT1aR complex. Morphologically, the heteromers were visualized by confocal FRET imaging at the cell surface and found have a role in modulating AT1aR trafficking. It was also found that the SCTR/AT1aR complex affected Gαs signaling specifically, reducing maximal response values by 24.3 ± 2.8 % compared to CHO-K1 cells transfected with only SCTR. While, this negative effect could be abolished by co-application of SCT and ANGII peptides, use of constitutively active AT1aR mutants or disruption of the hetero-complex using SCTR mutants. Taken together, the SCTR/AT1aR complex was proposed to impose conformational restraints on the SCTR that could be overcome upon activation of the AT1aR. Physiologically, hyperosmolality isovolemic induced drinking could be attenuated by central administration of TM peptides and the protein kinase A pathway blocker H-89, indicating receptor oligomerization to have a role in neural osmoregulation via a Gαs dependent pathway. This study presents novel findings regarding the receptor oligomerization of SCTR and AT1aR, which may be the molecular basis to the overlapping roles of SCT and ANGII in water homeostasis.