LAUSR

Fluorescence micrographs of the plasma membrane of cells expressing fluorescently labeled G Protein-Coupled Receptors (GPCRs) often exhibit small clusters of pixels (or puncta) with intensities that are higher than those of the surrounding pixels. While studies of GPCR interactions in uniform membrane areas abound, understanding the details of the GPCR interactions within such puncta as well as the nature of the membrane formations underlying the puncta is hampered by the lack of adequate experimental techniques. Here we introduce an enhancement of a recently developed method termed Fluorescence Intensity Fluctuation (FIF) spectrometry, which permits analysis of protein-protein interactions within the puncta in live cell membranes. We applied the novel FIF data analysis protocol to previously published data from cells expressing human secretin receptors (hSecR) and determined that the oligomer size increases with receptor concentration and duration of treatment with cognate ligand, not only within uniform regions of the membrane (in agreement with previous publications), but also within the puncta. In addition, we found that the number density and fractional area of the puncta increased following treatment with ligand. This method could be applied for probing the evolution in time of the chain of events that begins with ligand binding and continues with coated pits formation and receptor internalization for other GPCRs and, indeed, other membrane receptors in living cells.