The P2Y2 receptor promotes Wnt3a- and EGF-induced epithelial tubular formation by IEC6 cells by binding to integrins

Abstract

Epithelial tubular structures are essential units in various organs. Here, we used rat intestinal epithelial IEC6 cells to investigate tubulogenesis and we found that tubular formation was induced by a combination of Wnt3a and EGF signaling during threedimensional culture. Wnt3a and EGF induced the expression of the P2Y 2 receptor (P2Y 2 R, also known as P2RY2), and knockdown of P2Y 2 R suppressed tubular formation. A P2Y 2 R mutant that lacks nucleotide responsiveness rescued the phenotypes resulting from P2Y 2 R knockdown, suggesting that nucleotide-dependent responses are not required for P2Y 2 R functions in tubular formation. The ArgGly-Asp (RGD) sequence of P2Y 2 R has been shown to interact with integrins, and a P2Y 2 R mutant lacking integrin-binding activity was unable to induce tubular formation. P2Y 2 R expression inhibited the interaction between integrins and fibronectin, and induced cell morphological changes and proliferation. Inhibition of integrin and fibronectin binding by treatment with the cyclic RGD peptide and fibronectin knockdown induced tubular formation in the presence of EGF alone, but a fibronectin coat suppressed Wnt3a-and EGFinduced tubular formation. These results suggest that Wnt3a-and EGF-induced P2Y 2 R expression causes tubular formation by preventing the binding of integrins and fibronectin rather than by mediating nucleotide responses.

Palmitoylated CKAP4 regulates mitochondrial functions through an interaction with VDAC2 at ER–mitochondria contact sites

“…PLA was performed according to the manufacturer's protocol (Sigma) as described previously (Ibuka et al, 2015;Osugi et al, 2019). Cells transiently expressing FLAG-VDAC2 grown on glass coverslips were fixed for 15 min at RT in PBS containing 4% PFA.…”

P2 Receptors as Therapeutic Targets in the Salivary Gland: From Physiology to Dysfunction

“…Previous studies have demonstrated a role for the P2Y 2 R in corneal epithelial wound healing by increasing cell migration (Boucher et al, 2010), in liver regeneration by stimulating hepatocyte proliferation (Tackett et al, 2014), in cardiac regeneration by stimulating cardiac progenitor cell proliferation (Khalafalla F.G. et al, 2017) and in intestinal epithelial cell tubulogenesis (Ibuka et al, 2015). Activation of P2Y 2 Rs in the HSG cell line also induces the transactivation, homodimerization and autophosphorylation of the EGFR, a receptor tyrosine kinase known to be crucial for salivary gland branching morphogenesis and development (Miyazaki et al, 2004;Patel et al, 2006;Mizukoshi et al, 2016).…”

P2Y1‐like nucleotide receptors—Structures, molecular modeling, mutagenesis, and oligomerization

“…At the K289R mutant, the agonists ADP and UDP were more potent (four‐ and seven‐fold increased potency, respectively) than ATP and UTP (300‐ and 26‐fold decrease, respectively). Interestingly, the mutation R264L in the rat P2Y 2 R, which corresponds to Arg265 in the human receptor, had no impact on basal or ATPγS‐dependent activation, indicating that Arg264 in the rat receptor may not be involved in agonist‐induced signaling …”

“…Interestingly, the mutation R264L in the rat P2Y 2 R, which corresponds to Arg265 in the human receptor, had no impact on basal or ATPγS-dependent activation, indicating that Arg264 in the rat receptor may not be involved in agonist-induced signaling. 42 F I G U R E 9 Schematic tree diagram of available mutagenesis data for agonist and antagonist potencies of the human P2Y 2 R…”