Abstract
G-protein-coupled receptors (GPCRs) represent the largest and most diverse family of cell surface receptors. Several GPCRs have been documented to dimerize with resulting changes in pharmacology and signaling. We have previously reported, by means of photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence correlation spectroscopic analysis in live cells, that human somatostatin receptor (hSSTR) 5 could both homodimerize and heterodimerize with hSSTR1 in the presence of the agonist SST-14. By contrast, hSSTR1 remained monomeric when expressed alone regardless of agonist exposure in live cells. However, the effect of the agonist on other hSSTR members remains unknown. Using pbFRET microscopy and Western blot, we provide evidence for agonist-dependent dissociation of self-associated hSSTR2 stably expressed in CHO-K1 and HEK-293 cells. Furthermore, the dissociation of the hSSTR2 dimer occurred in a concentration-dependent manner. Moreover, blocking receptor dissociation using a cross-linker agent perturbed receptor trafficking. Taking these data together, we suggest that the process of GPCR dimerization may operate differently, even among members of the same family, and that receptor dissociation as well as dimerization may be important steps for receptor dynamics.
Highlights
G-protein-coupled receptors (GPCRs) represent the largest and most diverse family of cell surface receptors
By means of photobleaching fluorescence resonance energy transfer microscopy and fluorescence correlation spectroscopic analysis in live cells, that human somatostatin receptor 5 could both homodimerize and heterodimerize with hSSTR1 in the presence of the agonist SST-14
Cells were treated with different concentrations of SST-14 and processed for hSSTR2 localization to study by photobleaching fluorescence resonance energy transfer (pbFRET) microscopy. pbFRET microscopy was determined by the photobleaching decay of donor on the cell surface by using monoclonal anti-HA antibodies conjugated with either fluorescein or rhodamine
Summary
One model suggests that ligand binding induces a conformational change in the receptor that favors dimer formation In contrast to this model, the presence of GPCRs, which may be assembled as preformed dimers, has been shown for members of the class C subfamily, which includes GABAergic receptors (6 – 8), calcium-sensing receptor [9, 10], the metabotropic glutamate receptor [11], and the sweet taste receptors [12,13,14]. We show that agonist induced a dissociation of preassembled hSSTR2 dimers in a concentration-dependent manner This effect was inhibited when cell membranes were pretreated with a cross-linking agent. Dissociation of Somatostatin Receptor Dimers dependent dissociation of self-associated dimers may be a requirement for proper receptor trafficking
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