Oral Presentation Australian & New Zealand Obesity Society 2016 Annual Scientific Meeting

Signal transduction pathways activated by the orexigenic gut derived hormone insulin-like peptide 5 at relaxin family peptide receptor 4 (#54)

Dana S Hutchinson 1 , Sheng Y Ang 1 , Bronwyn A Evans 1 , Roger J Summers 1 , Martina Kocan 1
  1. Monash University, Parkville, VIC, Australia

Introduction. Insulin-like peptide 5 (INSL5) is a two-chain, three-disulphide bonded peptide belonging to the insulin/relaxin superfamily expressed in the enteroendocrine L-cells of human and mouse colon (Grosse et al, 2014). It is the cognate ligand for relaxin family peptide receptor 4 (RXFP4) a GPCR mainly expressed in the colorectum and enteric nervous system. Currently little is known of the signal transduction pathways activated by RXFP4.

Aims. This study examined intracellular signalling pathways activated by INSL5 acting at the human RXFP4 receptor stably expressed in CHO cells.

Methods. Cell signalling was investigated using AlphaScreen® assays. Ca2+ flux was monitored in a Flexstation® using X-rhod-1AM. RXFP4 recruitment of Gαi/o protein isoforms were determined by rescue of ERK1/2 responses by PTX-insensitive Gαi/o C351I mutants (mGαi/o). Cell proliferation was studied by bromo-deoxyuridine (BrdU) cell proliferation ELISA. RXFP4 interactions with β-arrestins 1/2, G protein-coupled receptor kinase 2 (GRK2), KRas and Rab5a were examined using real-time BRET.

Results. Mouse INSL5 inhibited forskolin-stimulated cAMP accumulation and activated ERK1/2, p38MAPK, Akt-Ser473, Akt-Thr308 and S6 ribosomal protein (S6RP) more potently than human INSL5. No Ca2+ mobilisation was observed.  PTX-abolished INSL5-stimulated ERK1/2 signal was rescued by mGαoA, mGαoB, mGαi2 and to a lesser extent by mGαi1 and mGαi3. RXFP4 interacted with GRK2, β-arrestins 1/2 and Rab5a but dissociated from KRas.  

Discussion. INSL5 negatively regulates cAMP production and activates multiple signalling pathways important for diverse cellular functions including growth, differentiation and proliferation (ERK1/2, p38MAPK, Akt) and protein synthesis (S6RP).  Following INSL5 activation, RXFP4 recruits a variety of Gαi/o and is regulated by β-arrestin 1/2 and GRK2 leading to receptor internalisation. Information on signalling pathways activated by INSL5 at RXFP4 is essential for understanding the biological roles of this novel gut hormone.        

 

Grosse et al (2014) PNAS. 111:33-38