Bradykinin sequesters B2 bradykinin receptors and the receptor-coupled gα subunits Gα(q) and Gα(i) in caveolae in DDT₁ MF-2 smooth muscle cells

In this report, we show that the vasoactive peptide agonist bradykinin (BK) when bound to B2 BK receptors on DDT₁ MF-2 smooth muscle cells promotes the recruitment and sequestration of the occupied receptors and the receptor- coupled G-protein α subunits Gα(q) and Gα(i) in caveolae. Association of ligand receptor complexes and Gα subunits with caveolae was indicated by their co-enrichment on density gradients with caveolin, a marker protein for caveolae. Caveolin and Gα subunits were monitored by immunoblotting, whereas receptors were monitored as ligand receptor complexes formed by labeling receptors with the agonist BK or the antagonist NPC17731 prior to cell disruption and caveolae enrichment. These complexes were detected with radioligand and by immunoblotting with BK antibodies. A direct interaction of Gα subunits with caveolin was also indicated by their co- immunoprecipitation. Immunoelectron microscopy revealed that the enriched caveolin, Gα subunits, and BK receptor complexes were present in structures of 0.1-0.2 μm. At 4 °C, BK and NPC17731 receptor complexes were detected in caveolae, and both complexes were sensitive to acid washing prior to cell disruption and caveolae enrichment. Elevation of the temperature to 37 °C increased the amount of BK receptor complexes in caveolae with a maximal response at 10 min (continuous labeling) or 20 min (single-round labeling), and the complexes became acid-resistant. These conditions also increased the amount of Gα(q) and Gα(i) in caveolae with a maximal response at 5-10 min. In contrast, the NPC17731 receptor complexes remained acid-sensitive and dissociated at this temperature, and antagonists did not increase the amount of Gα subunits in caveolae. These results show that some agonists that act through G-protein-coupled receptors promote the association of their receptors and receptor-coupled Gα subunits with caveolae.