Tetraspanins comprise a diverse family of four-pass transmembrane proteins that play critical roles in the immune, reproductive, genitourinary, and auditory systems. Despite their pervasive roles in human physiology, little is known about the structure of tetraspanins or the molecular mechanisms underlying their various functions. Here, we report the crystal structure of human CD81, a full-length tetraspanin. The transmembrane segments of CD81 pack as two largely separated pairs of helices, capped by the large extracellular loop (EC2) at the outer membrane leaflet. The two pairs of helices converge at the inner leaflet to create an intramembrane pocket with additional electron density corresponding to a bound cholesterol molecule within the cavity. Molecular dynamics simulations identify an additional conformation in which EC2 separates substantially from the transmembrane domain. Cholesterol binding appears to modulate CD81 activity in cells, suggesting a potential mechanism for regulation of tetraspanin function.

Cleavage of membrane-anchored proteins by ADAM (a disintegrin and metalloproteinase) endopeptidases plays a key role in a wide variety of biological signal transduction and protein turnover processes. Among ADAM family members, ADAM10 stands out as particularly important because it is both responsible for regulated proteolysis of Notch receptors and catalyzes the non-amyloidogenic α-secretase cleavage of the Alzheimer's precursor protein (APP). We present here the X-ray crystal structure of the ADAM10 ectodomain, which, together with biochemical and cellular studies, reveals how access to the enzyme active site is regulated. The enzyme adopts an unanticipated architecture in which the C-terminal cysteine-rich domain partially occludes the enzyme active site, preventing unfettered substrate access. Binding of a modulatory antibody to the cysteine-rich domain liberates the catalytic domain from autoinhibition, enhancing enzymatic activity toward a peptide substrate. Together, these studies reveal a mechanism for regulation of ADAM activity and offer a roadmap for its modulation.

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and β2AR, supporting the concept of β-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.

G protein-coupled receptors (GPCRs) can engage multiple pathways to activate ERK1/2 via both G proteins and/or ßarrestin. Receptor recruitment of ßarrestin is also important for GPCR desensitization, internalization and resensitization. Modulation of the receptor/ßarrestin interaction through modification of either component would presumably alter the output generated by receptor activation. Here we examined how ßarrestins regulate bradykinin (BK) B2 receptor (B2R) signalling and desensitization by either truncating ßarrestin1 or ßarrestin2 or by alanine substitution of a serine/threonine cluster in the C-terminal tail of B2R (B2R-4A), conditions which all affect the avidity of the B2R/ßarrestin complex. We first demonstrate that BK-mediated ERK1/2 activation is biphasic containing an early peak (between 2-5min) followed by sustained activation for at least 60min. The early but not the sustained phase was predictably affected by inhibition of either Gαq/11 or Gαi/o, whereas loss of ßarrestin2 but not ßarrestin1 resulted in diminished prolonged ERK1/2 activation. ßarrestin2's role was further examined using a truncation mutant with augmented avidity for the agonist-occupied receptor, revealing an increase in both immediate and extended ERK1/2 signalling. We also show that ERK1/2 is recruited to the B2R/ßarrestin complex on endosomes as well as the plasma membrane. Moreover, we investigated ßarrestin's role using the B2R-4A, which is deficient in ßarrestin binding and does not internalize. We show that ERK1/2 signalling downstream of the receptor is entirely G protein-dependent and receptor-mediated intracellular calcium mobilization studies revealed a lack of desensitization. Functionally, the lack of desensitization resulted in increased cell growth and migration compared to the wild-type receptor, which was sensitive to MEK inhibition. These results highlight ßarrestin's crucial role in the maintenance of proper B2R signalling.

Clathrin-mediated endocytosis is a complex process regulated at many different levels. We showed previously that activation of the angiotensin type 1 receptor (AT1R), which belongs to the G protein-coupled receptor (GPCR) family, leads to c-Src-dependent tyrosine phosphorylation of beta2-adaptin, a subunit of the clathrin adaptor AP-2. The phosphorylation of beta2-adaptin on tyrosine residue 737 (Y737) negatively regulates its interaction with betaarrestin, another important clathrin adaptor for GPCR internalization. Here we sought to determine whether AP-2 phosphorylation represents a general mechanism for different receptors internalizing through the clathrin pathway. Using a specifically designed antibody against the phosphorylated form of Y737 on beta2-adaptin, we demonstrate that this residue is phosphorylated by AT1R in different cell types like HEK293, COS-7 and vascular smooth muscle cells. Using RNA interference approaches, we reveal that this agonist-mediated event is both betaarrestin- and c-Src-dependent, and that it occurs at the plasma membrane in clathrin-coated vesicles (CCVs). We further show that this is not only a common event employed by other GPCRs like the beta2-adrenergic, vasopressin V2, bradykinin type 2, platelet-activating factor and endothelin A receptors but that the epidermal growth factor receptor is capable of eliciting the phosphorylation of AP-2 in CCVs. Our results imply that tyrosine phosphorylation of Y737 on beta2-adaptin is a common regulatory mechanism employed by different receptors undergoing clathrin-dependent endocytosis, and suggest a wider function for this event than originally anticipated.