BACKGROUND Biosynthesis of extragonadal androgen may contribute to the progression of castration-resistant prostate cancer. We evaluated whether abiraterone acetate, an inhibitor of androgen biosynthesis, prolongs overall survival among patients with metastatic castration-resistant prostate cancer who have received chemotherapy. METHODS We randomly assigned, in a 2:1 ratio, 1195 patients who had previously received docetaxel to receive 5 mg of prednisone twice daily with either 1000 mg of abiraterone acetate (797 patients) or placebo (398 patients). The primary end point was overall survival. The secondary end points included time to prostate-specific antigen (PSA) progression (elevation in the PSA level according to prespecified criteria), progression-free survival according to radiologic findings based on prespecified criteria, and the PSA response rate. RESULTS After a median follow-up of 12.8 months, overall survival was longer in the abiraterone acetate–prednisone group than in the placebo–prednisone group (14.8 months vs. 10.9 months; hazard ratio, 0.65; 95% confidence interval, 0.54 to 0.77; P<0.001). Data were unblinded at the interim analysis, since these results exceeded the preplanned criteria for study termination. All secondary end points, including time to PSA progression (10.2 vs. 6.6 months; P<0.001), progression-free survival (5.6 months vs. 3.6 months; P<0.001), and PSA response rate (29% vs. 6%, P<0.001), favored the treatment group. Mineralocorticoid-related adverse events, including fluid retention, hypertension, and hypokalemia, were more frequently reported in the abiraterone acetate–prednisone group than in the placebo–prednisone group. CONCLUSIONS The inhibition of androgen biosynthesis by abiraterone acetate prolonged overall survival among patients with metastatic castration-resistant prostate cancer who previously received chemotherapy. (Funded by Cougar Biotechnology; COU-AA-301 ClinicalTrials.gov number, NCT00638690.)

The ability of a system to regulate its responsiveness in the presence of a continuous stimulus, often termed desensitization, has been extensively characterized for the beta2-adrenergic receptor (beta2AR). beta2AR signalling is rapidly attenuated through receptor phosphorylation and subsequent binding of the protein beta-arrestin. Ultimately the receptor undergoes internalization, and although the molecular mechanism is unclear, receptor phosphorylation and beta-arrestin binding have been implicated in this processs. Here we report that beta-arrestin and arrestin-3, but not visual arrestin, promote beta2AR internalization and bind with high affinity directly and stoichiometrically to clathrin, the major structural protein of coated pits. Moreover, beta-arrestin/arrestin chimaeras that are defective in either beta2AR or clathrin binding show a reduced ability to promote beta2AR endocytosis. Immunofluorescence microscopy of intact cells indicates an agonist-dependent colocalization of the beta2AR and beta-arrestin with clathrin. These results show that beta-arrestin functions as an adaptor in the receptor-mediated endocytosis pathway, and suggest a general mechanism for regulating the trafficking of G-protein-coupled receptors.

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G protein-mediated signal transduction involves agonist activation of a seven transmembrane-spanning G protein-coupled receptor (GPR) 1 which, in turn, activates a heterotrimeric guanine nucleotide-binding protein (G protein). It is now well established that both the ␣ and ␤␥ subunits of G proteins modulate the activity of many effectors including adenylyl cyclases, phospholipases, ion channels, and cGMP phosphodiesterase (for review, see Refs. 1 and 2). Within seconds to minutes following agonist exposure, activated GPRs lose their ability to respond to agonist with their original sensitivity, a phenomenon commonly referred to as desensitization. Receptor desensitization is initiated by phosphorylation of the agonist-activated GPR by a family of enzymes known as G protein-coupled receptor kinases (GRKs) leading to high affinity binding of a second class of proteins known as arrestins (for review, see Refs. 3 and 4). It is thought that arrestin binding to the phosphorylated GPR sterically inhibits G protein binding (5). To date, four mammalian arrestins have been identified. These include two visual arrestins (arrestin and C-or X-arrestin) (6 -8), which likely regulate photoreceptors based on their restricted localization, and two nonvisual arrestins (␤-arrestin and arrestin3) (9 -12), which are ubiquitous and likely regulate a wide variety of GPRs.Another level of regulation of GPR signaling involves internalization of the activated receptor into a compartment distinct from the plasma membrane, a process known as sequestration (for review, see Ref. 13). Utilizing a variety of techniques, many studies have demonstrated that at least one mechanism by which GPRs internalize is via clathrin-coated pits (14 -19). Interestingly, recent studies have demonstrated a role for sequestration in resensitization of the activated GPR, suggesting a model in which the desensitized receptor is dephosphorylated by an intracellular vesicle-derived phosphatase and then recycled back to the plasma membrane (20 -23).Recently, several studies have implicated GRKs and arrestins in the internalization of agonist-activated GPRs. Overexpression of GRK2 (␤-adrenergic receptor kinase) enhanced sequestration of coexpressed m2 muscarinic acetylcholine receptor, whereas a dominant-negative ␤-adrenergic receptor kinase inhibited receptor sequestration (24). Similarly, overexpression of ␤-adrenergic receptor kinase rescued sequestration of a ␤ 2 -adrenergic receptor mutant (Y326A) defective in its ability to be sequestered, whereas sequestration of ␤ 2 -adrenergic receptor mutant Y326A lacking ␤-adrenergic receptor kinase phosphorylation sites was not rescued (25). Indeed, coexpression of either GRK3, GRK4, GRK5, or GRK6, but not the photoreceptor-specific GRK1, also enhanced sequestration of ␤ 2 -adrenergic mutant Y326A (26). It was further observed that overexpression of ␤-arrestin or arrestin3 (␤-arrestin2) alone rescued the sequestration of ␤ 2 -adrenergic mutant Y326A, whereas putative dominant-negative mutants of these arrestins inhibited ...

Receptor-mediated endocytosis is a pathway by which plasma membrane receptors are internalized selectively into cells through clathrin-coated pits (for review, see Refs. 1 and 2). Receptors are either internalized constitutively (e.g. some nutrient receptors) or internalized preferentially following ligand binding (e.g. some hormone receptors). Among those that belong to the latter category are several G protein-coupled receptors (GPRs).1 Many GPRs are phosphorylated rapidly upon agonist activation, and it is this phosphorylation event that leads to the selective binding of a family of proteins termed arrestins. To date there are four known arrestins: two visual arrestins (arrestin and cone arrestin) and two nonvisual arrestins (␤-arrestin and arrestin3; for review, see Ref.3). One role of arrestin binding is to uncouple the GPR from its cognate heterotrimeric G protein (desensitization), probably through direct competition with the G protein for the activated GPR (4). Following desensitization, the receptor is believed to be translocated into an intracellular compartment (5), a process commonly referred to as sequestration. Interestingly, sequestration also appears to be required for resensitization of receptors by allowing for receptor dephosphorylation and recycling (6, 7).Studies of the m2 muscarinic acetylcholine receptor (8) and a sequestration-deficient mutant form of the ␤ 2 -adrenergic receptor (9) have implicated GPR kinases and arrestins in GPR sequestration. Recent work from our laboratories has provided a plausible molecular mechanism for this internalization process, in which arrestins function as clathrin adaptors in the uptake of ␤ 2 -adrenergic (10) and other 2 GPRs. We found that nonvisual arrestins bind to clathrin with a K d of 10 -60 nM, comparable to that of the clathrin associated protein AP-2 (12, 13). Moreover, ␤ 2 -adrenergic receptor, ␤-arrestin, and clathrin colocalize in vivo upon agonist addition in an arrestin-dependent manner, indicating that the arrestin/clathrin interaction observed in vitro likely occurs in vivo in the presence of an activated receptor. These results suggest that the arrestin/ clathrin interaction is of central importance in regulating GPR trafficking.Clathrin, the major structural component of the coated pit lattice, consists of three heavy chains (M r Ϸ 192,000) and three light chains (M r Ϸ 26,000) (for review, see Ref. 14). By limited proteolysis of preformed clathrin cages, the heavy chain has been shown to comprise two distinct domains (15). An Ϸ50-kDa amino-terminal globular region, the terminal domain (TD), lies at the distal end of each triskelion leg. The remaining Ϸ140 kDa of the heavy chain forms the triskelion core, consisting of the vertex, proximal leg, and a portion of the distal leg of the clathrin trimer. One clathrin light chain is associated with each proximal leg. Based on electron microscopic studies and models of the polygonal lattice, a clathrin hub lies at each vertex, and each lattice edge is formed by the superposition of two distal a...

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