Distinct conformations of the corticotropin releasing factor type 1 receptor adopted following agonist and antagonist binding are differentially regulated.

The corticotropin releasing factor (CRF) type 1 receptor (CRF1) is a class B family G protein-coupled receptor that regulates the hypothalamic-pituitary-adrenal stress axis. Astressin is an amino-terminal truncated analog of CRF that retains high affinity binding to the extracellular domain of the receptor and is believed to act as a neutral competitive antagonist of receptor activation. Here we show that despite being unable to activate the CRF1 receptor, astressin binding results in the internalization of the receptor. Furthermore, entirely different pathways of internalization of CRF1 receptors are utilized following CRF and astressin binding. CRF causes the receptor to be phosphorylated, recruit beta-arrestin2, and to be internalized rapidly, likely through clathrin-coated pits. Astressin, however, fails to induce receptor phosphorylation or beta-arrestin2 recruitment, and internalization is slow and occurs through a pathway that is insensitive to inhibitors of clathrin-coated pits and caveolae. The fate of the internalized receptors also differs because only CRF-induced internalization results in receptor down-regulation. Furthermore, we present evidence that for astressin to induce internalization it must interact with both the extracellular amino terminus and the juxtamembrane domain of the receptor. Astressin binds with 6-fold higher affinity to full-length CRF1 receptors than to a chimeric protein containing only the extracellular domain attached to the transmembrane domain of the activin IIB receptor, yet two 12-residue analogs of astressin have similar affinities for both proteins but are unable to induce receptor internalization. These data demonstrate that agonists and antagonists for CRF1 receptors promote distinct conformations, which are then differentially regulated.

Vinculin modulation of paxillin-FAK interactions regulates ERK to control survival and motility.

Cells lacking vinculin are highly metastatic and motile. The reasons for this finding have remained unclear. Both enhanced survival and motility are critical to metastasis. Here, we show that vinculin null (vin-/-) cells and cells expressing a vinculin Y822F mutant have increased survival due to up-regulated activity of extracellular signal-regulated kinase (ERK). This increase is shown to result from vinculin's modulation of paxillin-FAK interactions. A vinculin fragment (amino acids 811-1066) containing the paxillin binding site restored apoptosis and suppressed ERK activity in vin-/- cells. Both vinY822F and vin-/- cells exhibit increased interaction between paxillin and focal adhesion kinase (FAK) and increased paxillin and FAK phosphorylation. Transfection with paxillin Y31FY118F dominant-negative mutant in these cells inhibits ERK activation and restores apoptosis. The enhanced motility of vin-/- and vinY822F cells is also shown to be due to a similar mechanism. Thus, vinculin regulates survival and motility via ERK by controlling the accessibility of paxillin for FAK interaction.

Differential desensitization, receptor phosphorylation, beta-arrestin recruitment, and ERK1/2 activation by the two endogenous ligands for the CC chemokine receptor 7.

Many members of the chemokine receptor family of G protein-coupled receptors utilize multiple endogenous ligands. However, differences between the signaling properties of multiple chemokines through a single receptor have yet to be well characterized. In this study we investigated the early signaling events of CCR7 initiated by its two endogenous ligands, CCL19 and CCL21. Both CCL19 and CCL21 induce G protein activation and calcium mobilization with equal potency. However, only activation by CCL19, not CCL21, promotes robust desensitization of endogenous CCR7 in the human T cell lymphoma cell line H9. Desensitization occurs through the induction of receptor phosphorylation and beta-arrestin recruitment (shown in HEK293 cells expressing CCR7-FLAG). The sites of CCL19-induced phosphorylation were mapped by mutating to alanines the serines and threonines found within kinase phosphorylation consensus sequences in the carboxyl terminus of CCR7. A cluster of sites, including Thr-373-376 and Ser-378 is important for CCL19-mediated phosphorylation of the receptor, whereas residues serine 356, 357, 364, and 365 are important for basal receptor phosphorylation by protein kinase C. Activation of CCR7 by both ligands leads to signaling to the ERK1/2 mitogen-activated protein kinase pathway. However, CCL19 promotes 4-fold more ERK1/2 phosphorylation than does CCL21. The mechanism by which CCL19 activates ERK1/2 was determined to be beta-arrestin-dependent, because it is reduced both by depletion of beta-arrestin-2 with small interfering RNA and by elimination of the phosphorylation sites in the tail of the receptor. Taken together, these findings demonstrate that CCL19 and CCL21 place CCR7 in functionally distinct conformations that are independent of their G protein-coupling potency: one that allows the efficient desensitization of the receptor and activation of ERK1/2, and another that is impaired in these functions.