Caveolin Interacts with the Angiotensin II Type 1 Receptor during Exocytic Transport but Not at the Plasma Membrane

Abstract

The mechanisms involved in angiotensin II type 1 receptor (AT 1 -R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT 1 -R is not concentrated in caveolae but is clustered in cholesterolindependent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT 1 -R in caveolae, AT 1 -R⅐caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT 1 -R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT 1 -R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT 1 -R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT 1 -R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT 1 -R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT 1 -R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT 1 -R through the exocytic pathway, but this does not result in AT 1 -R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT 1 -R.Lipid-based sorting mechanisms play an important role in the organization of the plasma membrane into microdomains (1-3). The biophysical properties of sphingolipids and cholesterol drive the spontaneous formation of lateral assemblies of liquid-ordered lipid rafts in a sea of liquid-disordered phospholipids. The biological importance of lipid rafts follows from the lateral segregation that they impose on membrane proteins. The differential distribution of plasma membrane proteins across raft and nonraft membranes in turn results in the concentration of specific groups of signaling proteins and lipids within discrete areas of the cell membrane (3-6). This increases the efficiency and specificity of signaling events by allowing more efficient interactions between proteins and by preventing cross-talk between different pathways.Caveolae are an abundant surface feature of many mammalian cells and represent a specific subtype of lipid raft. Functionally, caveolae have been implicated in endocytosis (7), potocytosis (8), transcytosis (9), apical transport (10), and cholesterol balance (11). Caveolae are identified by their characteristic morphology (flask-shaped, 55-65-nm diameter pits) and the presence of integral membrane proteins, termed caveolins, of which three mammalian isoforms have bee...

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