Angiotensin (Ang) (1-7) is the endogenous ligand for the G protein-coupled receptor Mas, a receptor (R) associated with cardiac, renal and cerebral protective responses. Physiological evidence suggests that Mas R undergoes agonist-dependent desensitization, but the underlying molecular mechanism regulating R activity is unknown. We investigated the hypothesis that Mas R desensitizes and internalizes upon stimulation with Ang-(1-7). For this purpose, we generated a chimera between the Mas R and the fluorescent protein YFP (MasR-YFP). MasR-YFP transfected HEK 293T cells were incubated with Ang-(1-7) and the relative cellular distribution of MasR-YFP was observed by confocal microscopy. In resting cells, MasR-YFP was mostly localized to the cell membrane. Ang-(1-7) induced a redistribution of MasR-YFP to intracellular vesicles of various sizes after 5 min. Following the time course of [125I]Ang-(1-7) endocytosis we observed that half of MasR-YFP underwent endocytosis after 10 min and this was blocked by a Mas R antagonist. MasR-YFP colocalized with Rab5, the early endosome antigen 1 and the adaptor protein complex 2, indicating that the R is internalized through a clathrin-mediated pathway and targeted to early endosomes after Ang-(1-7) stimulation. A fraction of MasR-YFP also colocalized with caveolin-1 suggesting that at some point MasR-YFP traverses caveolin-1 positive compartments. In conclusion, Mas R undergoes endocytosis upon stimulation with Ang-(1-7) and this event may explain the desensitization of Mas R responsiveness. In this way, Mas R activity and density may be tightly controlled by the cell.

The blue and green fluorescent proteins (BFP and GFP) have been fused at the N-and C-terminal ends, respectively, of the plasma membrane Ca 2؉ pump (PMCA) isoform 4xb (hPMCA4xb). The fusion protein was successfully expressed in yeast and purified by calmodulin affinity chromatography. Despite the presence of the fused autofluorescent proteins BFP-PMCA-GFP performed similarly to the wild-type enzyme with respect to Ca 2؉ -ATPase activity and sensitivity to calmodulin activation. In the autoinhibited state BFP-PMCA-GFP exhibited a significant intramolecular fluorescence resonance energy transfer (FRET) consistent with the location of the fluorophores at an average distance of 45 Å . The FRET intensity in BFP-PMCA-GFP decreased when the enzyme was activated either by Ca 2؉ -calmodulin, partial proteolysis, or acidic lipids. Moreover, FRET decreased and became insensitive to calmodulin when hPMCA4xb was activated by mutation D170N in BFP-PMCA(D170N)-GFP. The results suggest that the ends of the PMCA are in close proximity in the autoinhibited conformation, and they separate or reorient when the PMCA achieves its final activated conformation.The homeostasis of the intracellular Ca 2ϩ is crucial for cell function. The Ca 2ϩ ATPases from plasma membrane (PMCAs) 2 participate in the modulation of Ca 2ϩ signals and are responsible for the long term maintenance of the low concentration of intracellular Ca 2ϩ (1). The PMCAs belong to the P2-type ATPase superfamily of ion pumps and form an aspartyl phosphate intermediate during the transport cycle (2). Another essential feature of these ATPases is their ability to switch between two different conformational states from E 2 to E 1 in the presence of the transported ion.Human PMCAs are encoded by four separate genes, and additional variants are generated via alternative splicing of primary gene transcripts. PMCA4 is found virtually in all human tissues, and the splice variant xb is the most studied isoform. Computer modeling and sequence comparisons indicate that the overall structure of the PMCAs closely resembles that of other P-ATPases. Following the domain organization proposed for the SERCA (3, 4), the PMCA would contain a transmembrane region of 10 segments (M1-M10) and three major catalytic domains exposed to the cytosol. The nucleotide-binding (N) and the phosphorylation (P) domains contain the ATP binding site and the aspartate residue that forms the acyl phosphate intermediate, respectively, whereas the actuator domain (A) plays an essential role in the long range transmission of the conformational changes occurring during the transport cycle.Despite the clear overall homology, certain amino acid segments of the PMCA protein are not found in SERCA. The existence of these segments in the PMCA molecule has been generally associated with the extensive regulatory mechanisms that are known to alter the function of the PMCA. Indeed, the major difference between the two calcium pumps is the long C-terminal segment (C region) of the PMCA following M10, and this region is in...