Bradykinin sequesters B2 bradykinin receptors and the receptor-coupled G alpha subunits G alpha(q) and G alpha(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 alpha subunits G alpha(q) and G alpha(i) in caveolae, Association of ligand receptor complexes and G alpha subunits with caveolae was indicated by their co-enrichment on density gradients with caveolin, a marker protein for caveolae. Caveolin and G alpha 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 BR antibodies. A direct interaction of G alpha subunits with caveolin was also indicated by their co-immunoprecipitation, Immunoelectron microscopy revealed that the enriched caveolin, Ga subunits, and BK receptor complexes were present in structures of 0.1-0.2 mu m. At 4 degrees C, BH 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 degrees C increased the amount of BK receptor complexes in caveolae with a maximal response at 10 min (continuous labeling or 20 min (single-round labelling), and the complexes became acid-resistant, These conditions also increased the amount of G alpha(q), and G alpha(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 Get 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 Get subunits with caveolae.