Clathrin-mediated Endocytosis of m₃ Muscarinic Receptors

Abstract

Receptors as well as some G protein subunits internalize after agonist stimulation. It is not clear whether Gα_q or Gβγ undergo such regulated translocation. Recent studies demonstrate that m₃ muscarinic receptor activation in SK-N-SH neuroblastoma cells causes recruitment of tubulin to the plasma membrane. This subsequently transactivates Gα_q and activates phospholipase Cβ₁. Interaction of tubulin-GDP with Gβγ at the offset of phospholipase Cβ₁ signaling appears involved in translocation of tubulin and Gβγ to vesicle-like structures in the cytosol (Popova, J. S., and Rasenick, M. M. (2003) J. Biol. Chem. 278, 34299–34308). The relationship of this internalization to the clathrin-mediated endocytosis of the activated m₃ muscarinic receptors or Gα_q involvement in this process has not been clarified. To test this, SK-N-SH cells were treated with carbachol, and localization of Gα_q, Gβγ, tubulin, clathrin, and m₃ receptors were analyzed by both cellular imaging and biochemical techniques. Upon agonist stimulation both tubulin and clathrin translocated to the plasma membrane and co-localized with receptors, Gα_q and Gβγ. Fifteen minutes later receptors, Gβγ and tubulin, but not Gα_q, internalized with the clathrin-coated vesicles. Coimmunoprecipitation of m₃ receptors with Gβγ, tubulin, and clathrin from the cytosol of carbachol-treated cells was readily observed. These data suggested that Gβγ subunits might organize the formation of a multiprotein complex linking m₃ receptors to tubulin since they interacted with both proteins. Such protein assemblies might explain the dynamin-dependent but β-arrestin-independent endocytosis of m₃ muscarinic receptors since tubulin interaction with dynamin might guide or insert the complex into clathrin-coated pits. This novel mechanism of internalization might prove important for other β-arrestin-independent endocytic pathways. It also suggests cross-regulation between G protein-mediated signaling and the dynamics of the microtubule cytoskeleton.