Metastatic breast cancer cells have reduced calcium and actin response after ATP-P2Y2 signaling.

The tumor microenvironment and wound healing after injury, both contain extremely high concentrations of the extracellular signaling molecule, adenosine triphosphate (ATP) compared to normal tissue. P2Y2 receptor, an ATP-activated purinergic receptor, is typically associated with pulmonary, endothelial, and neurological cell signaling. Here we report its role and importance in breast epithelial cell signaling and how it’s altered in metastatic breast cancer. In response to ATP activation, P2Y2 receptor signaling causes an increase of intracellular Ca 2+ in non-tumorigenic breast epithelial cells, while their tumorigenic and metastatic counterparts have significantly reduced Ca 2+ responses. The non-tumorigenic cells respond to increased Ca 2+ with actin polymerization and localization to cellular junctions, while the metastatic cells remained unaffected. The increase in intracellular Ca 2+ after ATP stimulation could be blunted using a P2Y2 antagonist, which also prevented actin mobilization in non-tumorigenic breast epithelial cells. Furthermore, the lack of Ca 2+ concentration changes and actin mobilization in the metastatic breast cancer cells could be due to reduced P2Y2 expression, which correlates with poorer overall survival in breast cancer patients. This study elucidates rapid changes that occur after elevated intracellular Ca 2+ in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response. STATEMENT OF SIGNIFICANCE: This work shows non-tumorigenic breast epithelial cells increase intracellular Ca 2+ after ATP-P2Y2 signaling and re-localize actin, while metastatic cells lack this response, due to decreased P2Y2 expression, which correlates with poorer survival.

Conserved residues in the extracellular loop 2 regulate Stachel-mediated activation of ADGRG2.

Cleavage and dissociation of a large N-terminal fragment and the consequent unmasking of a short sequence (Stachel) remaining on the N-terminus have been proposed as mechanisms of activation of some members of the adhesion G protein-coupled receptor (aGPCR) family. However, the identity of residues that play a role in the activation of aGPCRs by the cognate Stachel remains largely unknown. Protein sequence alignments revealed a conserved stretch of residues in the extracellular loop 2 (ECL2) of all 33 members of the aGPCR family. ADGRG2, an orphan aGPCR, plays a major role in male fertility, Ewing sarcoma cell proliferation, and parathyroid cell function. We used ADGRG2 as a model aGPCR and generated mutants of the conserved residues in the ECL2 via site-directed mutagenesis. We show that tryptophan and isoleucine in the ECL2 are essential for receptor stability and surface expression in the HEK293 cells. By adjusting the receptor surface expression levels, we show that mutation of these residues of ECL2 ablates the Stachel-mediated activation of multiple signaling pathways of ADGRG2. This study provides a novel understanding of the role of the ECL2 in Stachel-mediated signaling and degradation of ADGRG2, which may lay the foundation for the rational design of therapeutics to target aGPCRs.

The Emerging Role of Adhesion GPCRs in Cancer.

Aberrant expression, function, and mutation of G protein-coupled receptors (GPCRs) and their signaling partners, G proteins, have been well documented in many forms of cancer. These cell surface receptors and their endogenous ligands are implicated in all aspects of cancer including proliferation, angiogenesis, invasion, and metastasis. Adhesion GPCRs (aGPCRs) form the second largest family of GPCRs, most of which are orphan receptors with unknown physiological functions. This is mainly due to our limited insight into their structure, natural ligands, signaling pathways, and tissue expression profiles. Nevertheless, recent studies show that aGPCRs play important roles in cell adhesion to the extracellular matrix and cell-cell communication, processes that are dysregulated in cancer. Emerging evidence suggests that aGPCRs are implicated in migration, proliferation, and survival of tumor cells. We here review the role of aGPCRs in the five most common types of cancer (lung, breast, colorectal, prostate, and gastric) and emphasize the importance of further translational studies in this field.