Abstract
G-protein coupled receptors (GPCRs) are known to form homo- and hetero- oligomers which are considered critical to modulate their function. However, studying the existence and functional implication of these complexes is not straightforward as controversial results are obtained depending on the method of analysis employed. Here, we use a quantitative single molecule super-resolution imaging technique named qPAINT to quantify complex formation within an example GPCR. qPAINT, based upon DNA-PAINT, takes advantage of the binding kinetics between fluorescently labelled DNA imager strands to complementary DNA docking strands coupled to protein targeting antibodies to quantify the protein copy number in nanoscale dimensions. We demonstrate qPAINT analysis via a novel pipeline to study the oligomerization of the purinergic receptor Y2 (P2Y2), a rhodopsin-like GPCR, highly expressed in the pancreatic cancer cell line AsPC-1, under control, agonistic and antagonistic conditions. Results reveal that whilst the density of P2Y2 receptors remained unchanged, antagonistic conditions displayed reduced percentage of oligomers, and smaller numbers of receptors in complexes. Yet, the oligomeric state of the receptors was not affected by agonist treatment, in line with previous reports. Understanding P2Y2 oligomerization under agonistic and antagonistic conditions will contribute to unravelling P2Y2 mechanistic action and therapeutic targeting.
Highlights
To demonstrate the potential of qPAINT to study G-protein coupled receptors (GPCRs) oligomerization status, we investigated the nanoscale distribution of the P2Y2 receptor, a member of the d subgroup of the family A of GPCRs, in the cancer cell line AsPC-1, as it endogenously expresses high levels of this receptor and single cells are imaged due to its low levels of cell grouping [19]
total internal reflection (TIR) excitation allows investigation of samples at or near the cell membrane by optically sectioning light illumination to only the most superficial ~100 nm of the sample. This is extremely beneficial in the study of GPCRs located at the plasma membrane as the receptors are
Optical microscopy techniques have Analysis been applied to the and molecular brightness approaches [13,14] were developed and applied to study a study of GPCRvariety oligomerization, with one of the first single molecule imaging studies done of GPCRs
Summary
G-protein coupled receptors (GPCRs) are the largest family of cell surface receptors in eukaryotic cells. These seven-transmembrane receptors have influence in physiological events such as cell to cell communication, immune responses, nerve transmission and even hunger and sleep regulation [1,2,3]. The role of GPCRs in diseases such as rheumatoid arthritis, heart disease, cancer, obesity, and neurodegenerative disorders accentuates the need to investigate this family of receptors further. More than a third of all drugs approved by the FDA target GPCRs [4] but often such drugs have a variety of poorly understood mechanisms, as a recent example surrounding opioid receptor agonists illustrates [5,6,7,8]
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