Emerging findings disclose unexpected components of G protein-coupled receptor (GPCR) signaling and cell biology. Select GPCRs exhibit classical signaling, that is restricted to cell membranes, as well as newly described persistent signaling that depends on internalization of the GPCR bound to β-arrestins. Termination of non-canonical endosomal signaling requires intraluminal acidification and sophisticated protein trafficking machineries. Recent studies reveal the structural determinants of the trafficking chaperones. This review summarizes advances in GPCR signaling and trafficking with a focus on the parathyroid hormone receptor (PTHR) as a prototype, and on the actin-sorting nexin 27 (SNX27)-retromer tubule (ASRT) complex, an endosomal sorting hub responsible for recycling and preservation of cell surface receptors. The findings are integrated into a model of PTHR trafficking with implications for signal transduction, bone growth, and mineral ion metabolism.

Many G protein-coupled receptors (GPCR) exert patterns of cell-specific signaling and function. Mounting evidence now supports the view that cytoplasmic adapter proteins contribute critically to this behavior. Adapter proteins recognize highly conserved motifs such as those for Src homology 3 (SH3), phosphotyrosine-binding (PTB), and postsynaptic density 95/discs-large/zona occludens (PDZ) docking sequences in candidate GPCRs. Here we review the behavior of the Na⁺/H⁺ exchange regulatory factor (NHERF) family of PDZ adapter proteins on GPCR signalling, trafficking, and function. Structural determinants of NHERF proteins that allow them to recognize targeted GPCRs are considered. NHERF1 and NHERF2 are capable also of modifying the assembled complex of accessory proteins such as β-arrestins, which have been implicated in regulating GPCR signaling. In addition, NHERF1 and NHERF2 modulate GPCR signaling by altering the G protein to which the receptor binds or affect other regulatory proteins that affect GTPase activity, protein kinase A, phospholipase C, or modify downstream signaling events. Small molecules targeting the site of NHERF1-GPCR interaction are being developed and may become important and selective drug candidates.

Bone growth and remodeling depend upon the opposing rates of bone formation and resorption. These functions are regulated by intrinsic seven transmembrane-spanning receptors, the parathyroid hormone receptor (PTH1R) and frizzled (FZD), through their respective ligands, parathyroid hormone (PTH) and Wnt. FZD activation of canonical beta-catenin signaling requires the adapter protein Dishevelled (Dvl). We identified a Dvl-binding motif in the PTH1R. Here, we report that the PTH1R activates the beta-catenin pathway by directly recruiting Dvl, independent of Wnt or LRP5/6. PTH1R coimmunoprecipitated with Dvl. Deleting the carboxyl-terminal PTH1R PDZ-recognition domain did not abrogate PTH1R-Dvl interactions; nor did truncating the receptor at position 480. However, further deletion eliminating the putative Dvl recognition domain abolished PTH1R interactions with Dvl. PTH activated beta-catenin in a time- and concentration-dependent manner and translocated beta-catenin to the nucleus. beta-Catenin activation was inhibited by Dvl2 dominant negatives and by short hairpin RNA sequences targeted against Dvl2. PTH-induced osteoclastogenesis was also inhibited by Dvl2 dominant negative mutants. These findings demonstrate that G protein-coupled receptors other than FZD directly activate beta-catenin signaling, thereby mimicking many of the functions of the canonical Wnt-FZD pathway. The distinct modes whereby FZD and PTH1R activate beta-catenin control convergent or divergent effects on osteoblast differentiation, and osteoclastogenesis may arise from PTH1R-induced second messenger phosphorylation.

G protein-coupled receptors (GPCRs) are the largest and most diverse superfamily of membrane proteins and mediate most cellular responses to hormones and neurotransmitters. Posttranslational modifications are considered the main regulators of all GPCRs. In addition to phosphorylation, glycosylation, and palmitoylation, increasing evidence as reviewed here reveals that ubiquitination also regulates the magnitude and temporospatial aspects of GPCR signaling. Posttranslational protein modification by ubiquitin is a key molecular mechanism governing proteins degradation. Ubiquitination mediates the covalent conjugation of ubiquitin, a highly conserved polypeptide of 76 amino acids, to protein substrates. This process is catalyzed by 3 enzymes acting in tandem: an E1, ubiquitin-activating enzyme; an E2, ubiquitin-carrying enzyme; and an E3, ubiquitin ligase. Ubiquitination is counteracted by deubiquitinating enzymes that deconjugate ubiquitin-modified proteins and rescue the substrate from proteasomal degradation. Although ubiquitination is known to target many GPCRs for lysosomal or proteasomal degradation, emerging findings define novel roles for the basal status of ubiquitination and for rapid deubiquitination and transubiquitination controlling cell surface expression and cellular responsiveness of some GPCRs. In this review, we highlight the classical and novel roles of ubiquitin in the regulation of GPCR function, signaling, and trafficking.

The Na/H exchanger regulatory factors, NHERF1 and NHERF2, are adapter proteins involved in targeting and assembly of protein complexes. The parathyroid hormone receptor (PTHR) interacts with both NHERF1 and NHERF2. The NHERF proteins toggle PTHR signaling from predominantly activation of adenylyl cyclase in the absence of NHERF to principally stimulation of phospholipase C when the NHERF proteins are expressed. We hypothesized that this signaling switch occurs at the level of the G protein. We measured G protein activation by [(35)S]GTPgammaS binding and G(alpha) subtype-specific immunoprecipitation using three different cellular models of PTHR signaling. These studies revealed that PTHR interactions with NHERF1 enhance receptor-mediated stimulation of G(alpha)(q) but have no effect on stimulation of G(alpha)(i) or G(alpha)(s). In contrast, PTHR associations with NHERF2 enhance receptor-mediated stimulation of both G(alpha)(q) and G(alpha)(i) but decrease stimulation of G(alpha)(s). Consistent with these functional data, NHERF2 formed cellular complexes with both G(alpha)(q) and G(alpha)(i), whereas NHERF1 was found to interact only with G(alpha)(q). These findings demonstrate that NHERF interactions regulate PTHR signaling at the level of G proteins and that NHERF1 and NHERF2 exhibit isotype-specific effects on G protein activation.