STRL33/BONZO/TYMSTR is an orphan chemokine and HIV/SIV coreceptor receptor that is expressed on activated T lymphocytes. We describe an expression cloning strategy whereby we isolated a novel chemokine, which we name CXCL16. CXCL16 is an α (CXC) chemokine but also has characteristics of CC chemokines and a structure similar to fractalkine (neurotactin) in having a transmembrane region and a chemokine domain suspended by a mucin-like stalk. A recombinant version of CXCL16 fails to mediate chemotaxis to all known chemokine receptor transfectants tested but does mediate robust chemotaxis, high affinity binding, and calcium mobilization to Bonzo receptor transfectants, indicating that this is a unique receptor ligand interaction. In vitro polarized T cell subsets including Th1, Th2, and Tr1 cells express functional Bonzo, suggesting expression of this receptor in chronic inflammation, which we further verified by demonstration of CXCL16-mediated migration of tonsil-derived CD4+ T lymphocytes. CXCL16 is expressed on the surface of APCs including subsets of CD19+ B cells and CD14+ monocyte/macrophages, and functional CXCL16 is also shed from macrophages. The combination of unique structural features of both Bonzo and CXCL16 suggest that this interaction may represent a new class of ligands for this receptor family. Additionally, this chemokine might play a unique dual role of attracting activated lymphocyte subsets during inflammation as well as facilitating immune responses via cell-cell contact.

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.

The DRY motif is a triplet amino acid sequence (aspartic acid, arginine, and tyrosine) that is highly conserved in G protein-coupled receptors (GPCRs). Recently, we have shown that a molecular determinant for nephrogenic diabetes insipidus, the vasopressin receptor with a substitution at the DRY motif arginine (V2R R137H), is a constitutively desensitized receptor that is unable to couple to G proteins due to its constitutive association with beta-arrestin [Barak, L. S. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 93-98]. Additionally, the mutant receptors are localized in endocytic vesicles, identical to wild-type receptors stimulated with agonist. In this study, we asked whether the constitutively desensitized phenotype observed in the V2R R137H represents a general paradigm that may be extended to other GPCRs. We show that arginine substitutions in the DRY motifs of the alpha(1B) adrenergic receptor (alpha(1B)-AR) and angiotensin II type 1A receptor (AT(1A)R) result in receptors that are uncoupled from G proteins, associated with beta-arrestins, and found localized in endocytic vesicles rather than at the plasma membrane in the absence of agonists. The localization of the alpha(1B)-ARs and AT(1A)Rs with arginine substitutions can be restored to the plasma membrane by either using selective antagonists or preventing the endocytosis of the beta-arrestin-receptor complexes. These results indicate that the arginine residue of the DRY motif is essential for preserving the localization of the inactive receptor complex. Furthermore, constitutive desensitization may underlie some loss-of-function receptor phenotypes and represent an unappreciated mechanism of hormonal resistance.

The TEL/PDGF␤R gene, which encodes a fusion protein containing the ETS-family member TEL fused to the protein-tyrosine kinase domain of the platelet-derived growth factor receptor-␤ (PDGF␤R), confers interleukin 3 (IL-3)-independent growth on Ba/F3 hematopoietic cells. TEL/ PDGF␤R mutants have been generated that contain tyrosine-to-phenylalanine (Tyr3Phe) substitutions at phosphorylation sites present in the native PDGF␤R to assess the role of these sites in cell transformation by TEL/PDGF␤R. Similar to previous findings in a murine bone marrow transplantation model, full transformation of Ba/F3 cells to IL-3-independent survival and proliferation required the TEL/PDGF␤R juxtamembrane and carboxy terminal phosphorylation sites. In contrast to previous reports concerning comparable mutants in the native PDGF␤R, each of the TEL/PDGF␤R mutants is fully active as a protein-tyrosine kinase. IntroductionChronic myelomonocytic leukemia (CMML) is characterized by clonal proliferation of myeloid cells and frequent progression to acute leukemia. A recurring cytogenetic abnormality in CMML is the t(5;12)(q33;p13) chromosomal translocation. The resulting gene rearrangement fuses the 5Ј region of TEL to the gene encoding the platelet-derived growth factor receptor-␤ (PDGF␤R). 1 TEL is a member of the ETS family of transcription factors, and it consists of an amino terminal Pointed domain (PNT) and a carboxy terminal DNA-binding domain that shares sequence homology with the winged helix-turn-helix motif. In the TEL/PDGF␤R fusion protein, the amino terminal region of TEL (which contains the PNT domain) is fused to the membrane-spanning segment and the entire cytoplasmic protein-tyrosine kinase domain of PDGF␤R. The PNT domain present in the TEL segment mediates homotypic oligomerization of the fusion protein. 2,3 Fusion of TEL sequences to PDGF␤R results in constitutive oligomerization and activation of protein-tyrosine kinase activity 2 ; numerous examples of receptor tyrosine kinase activation through homotypic oligomerization have been described (reviewed by Lemmon and Schlessinger 4 ).The Ba/F3 murine hematopoietic cell line, which requires interleukin-3 (IL-3) for survival, 5 can be transformed to IL-3 independence by the TEL/PDGF␤R fusion. 2 TEL/PDGF␤R is constitutively phosphorylated on tyrosine residues, and a point mutation corresponding to a kinase-inactivating mutation in the native PDGF␤R abrogates TEL/ PDGF␤R kinase activation and transformation of Ba/F3 cells. Moreover, the PDGF␤R tyrosine kinase inhibitor CGP 57148 6 inhibits TEL/PDGF␤R kinase activity in vitro, inhibits autophosphorylation in vivo, and abrogates proliferation of Ba/F3 cells transformed by TEL/ PDGF␤R. 7 Thus, hematopoietic transformation by TEL/PDGF␤R requires protein-tyrosine kinase activity.Similar structure-function relationships have been described for the HIP1/PDGF␤R fusion 8,9 associated with the t(5;7)(q33;q11.2) translocation, and the H4/PDGF␤R fusion that is expressed as a consequence of the t(5;10)(q33;q22) translocation. 10 Each of...

GPCRs are a large family of cell-surface proteins that regulate many important biochemical pathways and physiological responses. The isolation and characterization of GPCRs represent one of the more remarkable success stories that occurred during the revolution in biology of the last quarter century. Of the many discoveries that originated in the laboratory of Robert Lefkowitz at Duke University concerning GPCR regulation, none is more fundamental than the elucidation of the families of GRKs and arrestin proteins that terminate GPCR signaling. In this essay, we will discuss how advances in microscopy and biology have made the visualization of GPCR, GRK, and arrestin activity possible in single cells. Additionally, we will discuss how imaging studies using arrestins and a naturally occurring mutant of the vasopressin receptor led to the recognition of a novel phenotypic receptor behavior, in which the receptor desensitizes in the absence of agonist. We have termed this process constitutive desensitization, and this unexpected receptor property suggests that it may be possible to develop novel classes of signal-inhibiting drugs distinct from conventional antagonists.

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