Grk5l controls heart development by limiting mTOR signaling during symmetry breaking.

The correct asymmetric placement of inner organs is termed situs solitus and is determined early during development. Failure in symmetry breaking results in conditions ranging from randomized organ arrangement to a complete mirror image, often accompanied by severe congenital heart defects (CHDs). We found that the zebrafish homolog of mammalian G protein-coupled receptor kinase 5 (GRK5) employs noncanonical, receptor-independent functions to secure symmetry breaking. Knockdown of GRK5's closest homolog in zebrafish embryos, Grk5l, is sufficient to randomize cardiac looping and left-right asymmetry. Mechanistically, we found that loss of GRK5 increases mammalian target of rapamycin complex 1 (mTORC1) activity. This causes elongation of motile cilia in the organ of laterality, a consequence that is known to be sufficient to trigger aberrant organ arrangement. By fine-tuning mTORC1, GRK5 thus serves an unanticipated function during early development, besides its well-characterized role in the adult heart. These findings could implicate GRK5 as a susceptibility allele for certain cases of CHD.

Smoothened signaling in vertebrates is facilitated by a G protein-coupled receptor kinase.

Smoothened, a heptahelical membrane protein, functions as the transducer of Hedgehog signaling. The kinases that modulate Smoothened have been thoroughly analyzed in flies. However, little is known about how phosphorylation affects Smoothened in vertebrates, mainly, because the residues, where Smoothened is phosphorylated are not conserved from Drosophila to vertebrates. Given its molecular architecture, Smoothened signaling is likely to be regulated in a manner analogous to G protein-coupled receptors (GPCRs). Previously, it has been shown, that arrestins and GPCR kinases, (GRKs) not only desensitize G protein-dependent receptor signaling but also function as triggers for GPCR trafficking and formation of signaling complexes. Here we describe that a GRK contributes to Smoothened-mediated signaling in vertebrates. Knockdown of the zebrafish homolog of mammalian GRK2/3 results in lowered Hedgehog transcriptional responses, impaired muscle development, and neural patterning. Results obtained in zebrafish are corroborated both in cell culture, where zGRK2/3 phosphorylates Smoothened and promotes Smoothened signal transduction and in mice where deletion of GRK2 interferes with neural tube patterning. Together, these data suggest that a GRK functions as a vertebrate kinase for Smoothened, promoting Hedgehog signal transduction during early development.

N-terminal tyrosine modulation of the endocytic adaptor function of the beta-arrestins.

The highly homologous beta-arrestin1 and -2 adaptor proteins play important roles in the function of G protein-coupled receptors. Either beta-arrestin variant can function as a molecular chaperone for clathrin-mediated receptor internalization. This role depends primarily upon two distinct, contiguous C-terminal beta-arrestin motifs recognizing clathrin and the beta-adaptin subunit of AP2. However, a molecular basis is lacking to explain the different endocytic efficacies of the two beta-arrestin isoforms and the observation that beta-arrestin N-terminal substitution mutants can act as dominant negative inhibitors of receptor endocytosis. Despite the near identity of the beta-arrestins throughout their N termini, sequence variability is present at a small number of residues and includes tyrosine to phenylalanine substitutions. Here we show that corresponding N-terminal (Y/F)VTL sequences in beta-arrestin1 and -2 differentially regulate mu-adaptin binding. Our results indicate that the beta-arrestin1 Tyr-54 lessens the interaction with mu-adaptin and moreover is a Src phosphorylation site. A gain of endocytic function is obtained with the beta-arrestin1 Y54F substitution, which improves both the beta-arrestin1 interaction with mu-adaptin and the ability to enhance beta2-adrenergic receptor internalization. These data indicate that beta-arrestin2 utilizes mu-adaptin as an endocytic partner, and that the inability of beta-arrestin1 to sustain a similar degree of interaction with mu-adaptin may result from coordination of Tyr-54 by neighboring residues or its modification by Src kinase. Additionally, these naturally occurring variations in beta-arrestins may also differentially regulate the composition of the signaling complexes organized on the receptor.

Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2.

Deregulation of the Sonic hedgehog pathway has been implicated in an increasing number of human cancers. In this pathway, the seven-transmembrane (7TM) signaling protein Smoothened regulates cellular proliferation and differentiation through activation of the transcription factor Gli. The activity of mammalian Smoothened is controlled by three different hedgehog proteins, Indian, Desert, and Sonic hedgehog, through their interaction with the Smoothened inhibitor Patched. However, the mechanisms of signal transduction from Smoothened are poorly understood. We show that a kinase which regulates signaling by many "conventional" 7TM G-protein-coupled receptors, G protein-coupled receptor kinase 2 (GRK2), participates in Smoothened signaling. Expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription. Moreover, knockdown of endogenous GRK2 by short hairpin RNA (shRNA) significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2. We find that GRK2 promotes the association between active Smoothened and beta-arrestin 2. Indeed, Gli-dependent signaling, mediated by coexpression of Smoothened and GRK2, is diminished by beta-arrestin 2 knockdown with shRNA. Together, these data suggest that GRK2 plays a positive role in Smoothened signaling, at least in part, through the promotion of an association between beta-arrestin 2 and Smoothened.

Beta-arrestin 2 regulates zebrafish development through the hedgehog signaling pathway.

beta-arrestins are multifunctional proteins that act as scaffolds and transducers of intracellular signals from heptahelical transmembrane-spanning receptors (7TMR). Hedgehog (Hh) signaling, which uses the putative 7TMR, Smoothened, is established as a fundamental pathway in development, and unregulated Hh signaling is associated with certain malignancies. Here, we show that the functional knockdown of beta-arrestin 2 in zebrafish embryos recapitulates the many phenotypes of Hh pathway mutants. Expression of wild-type beta-arrestin 2, or constitutive activation of the Hh pathway downstream of Smoothened, rescues the phenotypes caused by beta-arrestin 2 deficiency. These results suggest that a functional interaction between beta-arrestin 2 and Smoothened may be critical to regulate Hh signaling in zebrafish development.

Structural analysis of yoked chorionic gonadotropin-luteinizing hormone receptor ectodomain complexes by circular dichroic spectroscopy.

Binding of the heterodimeric glycoprotein hormone, chorionic gonadotropin (CG), occurs to the heptahelical LH receptor N-terminal ectodomain (ECD), a large portion of which has been modeled as a leucine-rich repeat protein. In this study, we expressed and purified three single chain N-CG-ECD-C complexes, one comprising the full-length ECD, 1-341 (encoded by exons 1-10 and a portion of 11), and two C-terminal ECD deletion fragments, 1-294 (encoded by exons 1-10) and 1-180 (encoded by exons 1-7). The fusion proteins, including yoked CG (N-beta-alpha-C), were characterized by Western blot analysis and circular dichroism (CD). Analysis of the CD spectra obtained on the CG-ECD fusion proteins, and of the difference spectrum of each after subtracting the CG contribution, yielded secondary structures consistent with a repeating beta-strand/alpha-helix fold as predicted in the homology model. A marked decrease in helicity was observed when the C-terminal 47 amino acid residues were removed from the ECD. Removal of an additional 114 residues, i.e. the region encoded by exons 8-10, results in the loss of fewer helical residues. These results suggest that the hinge region of the ECD, predicted to contain only limited secondary structure, interacts with and stabilizes the ligand-occupied N-terminal portion. Furthermore, the results support a repeating fold, consistent with the proposed model for the LHR ECD.

Consequences of single-chain translation on the structures of two chorionic gonadotropin yoked analogs in alpha-beta and beta-alpha configurations.

Human chorionic gonadotropin (hCG) is a placental-derived heterodimeric glycoprotein hormone, which, through the binding and activation of the LH receptor, rescues the corpus luteum and maintains pregnancy. The three-dimensional structure of hCG is known; however, the relevance of its fold to bioactivity is unclear. Although both subunits (alpha and beta) are required for activity, recent data with single-chain analogs have suggested a diminished role for the cystine knot and an intact heterodimeric interface in binding and receptor activation in vitro. Herein, we report the purification and structural characterization of two yoked (Y) hCG analogs, YhCG1 (beta-alpha) and YhCG3 (alpha-beta). The fusion proteins yielded higher IC50s and EC50s than those of hCG; the maximal hCG-mediated cAMP production, however, was the same. Circular dichroic spectroscopy revealed that the three proteins exhibit distinct far UV circular dichroic spectra, with YhCG1 containing somewhat more secondary structure than YhCG3 and hCG. Limited proteolysis with proteinase K indicated that heterodimeric hCG was much more resistant to cleavage than the single-chain analogs. YhCG1 was more susceptible to proteolysis than YhCG3, and the fragmentation patterns were different in the two proteins. Taken together, the data presented herein provide direct structural evidence for altered three-dimensional conformations in the two single-chain hCG analogs. Thus, the cognate G protein-coupled receptor can recognize and functionally respond to multiple ligand conformations.