Feedback inhibition of G protein-coupled receptor kinase 2 (GRK2) activity by extracellular signal-regulated kinases.

G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and beta-arrestin binding uncouple G protein-coupled receptors (GPCRs) from their respective G proteins and initiates the process of receptor internalization. In the case of the beta(2)-adrenergic receptor and lysophosphatidic acid receptor, these processes can lead to ERK activation. Here we identify a novel mechanism whereby the activity of GRK2 is regulated by feedback inhibition. GRK2 is demonstrated to be a phosphoprotein in cells. Mass spectrometry and mutational analysis localize the site of phosphorylation on GRK2 to a carboxyl-terminal serine residue (Ser(670)). Phosphorylation at Ser(670) impairs the ability of GRK2 to phosphorylate both soluble and membrane-incorporated receptor substrates and dramatically attenuates Gbetagamma-mediated activation of this enzyme. Ser(670) is located in a peptide sequence that conforms to an ERK consensus phosphorylation sequence, and in vitro, in the presence of heparin, ERK1 phosphorylates GRK2. Inhibition of ERK activity in HEK293 cells potentiates GRK2 activity, whereas, conversely, ERK activation inhibits GRK2 activity. The discovery that ERK phosphorylates and inactivates GRK2 suggests that ERK participates in a feedback regulatory loop. By negatively regulating GRK-mediated receptor phosphorylation, beta-arrestin-mediated processes such as Src recruitment and clathrin-mediated internalization, which are required for GPCR-mediated ERK activation, are inhibited, thus dampening further ERK activation.

Beta-adrenergic regulation of cholecystokinin secretion in STC-1 cells.

Previously, it has been shown that an increase in adenosine 3',5'-cyclic monophosphate (cAMP) levels stimulates intestinal secretion of cholecystokinin (CCK); however, the mechanisms for increasing intracellular cAMP levels are not known. Using the CCK-secreting intestinal cell line, STC-1, we evaluated whether beta-adrenergic receptors (beta-ARs) might be present on STC-1 cells and whether they stimulated CCK release through increases in cAMP. Photoaffinity labeling of beta-ARs from solubilized STC-1 cell membranes revealed photoincorporation of the agonist [125I]iodocyanopindolol into an approximately 75-kDa band. Addition of the beta-AR agonist, isoproterenol, in the presence of 3-isobutyl-1-methylxanthine, produced a concentration-dependent increase in both cAMP levels and CCK release. Blockade of beta 1- and/or beta 2-ARs significantly inhibited isoproterenol-stimulated increases in cAMP production and CCK release. With the use of fura 2-loaded cells to measure changes in intracellular Ca2+ concentration ([Ca2+]i), isoproterenol stimulation was found to increase cytosolic Ca2+ levels. To evaluate whether this increase in [Ca2+]i was due to release of Ca2+ or influx of Ca2+, cells were treated with the L-type calcium channel blocker, diltiazem, which inhibited isoproterenol-stimulated CCK secretion. Furthermore, in patch-clamp studies with inside-out membrane patches, addition of the catalytic subunit of protein kinase A activated diltiazem-sensitive Ca2+ channels. It is concluded that beta-ARs are present on STC-1 cells and are coupled to the production of cAMP, which may increase CCK release through a calcium-dependent process.

Differences in the cell surface antigens of prolactin-producing cells of human decidual and pituitary tissues.

Anterior pituitary cells and other endocrine cells which synthesize and release protein hormones share the cell surface ganglioside antigens A2B5 and 3G5. In addition, these peptide hormone-producing cells contain chromogranin, a major protein component of secretory vesicles. Since human decidual cells synthesize and release a PRL that has chemical and biological properties indistinguishable from those of pituitary PRL, we examined by immunochemical techniques whether decidual cells also contain cell surface antigens A2B5, 3G5, and chromogranin. In these studies, human decidual tissue from three pregnancies and human and rat pituitary tissues were processed and stained with mouse monoclonal antibodies to A2B5, 3G5, and chromogranin by indirect immunofluorescence using fluorescein-conjugated sheep antimouse gamma-globulin. In all instances, the pituitary and pancreatic tissue stained strongly with the monoclonal antibodies to the cell surface antigens, but the decidual tissues from the three pregnancies did not. The pituitary and pancreatic cells also stained strongly with the monoclonal antibody (LK 2H10) to chromogranin, but the decidual tissues had no reactivity. In control experiments, the decidual tissues stained strongly with a monoclonal antibody to HLA antigens, and none of the tissues stained with nonimmune gamma-globulin. These results strongly suggest that the PRL-producing cells of human decidua and pituitary have different cell surface antigens. The observation that decidual cells do not contain chromogranin is consistent with previous biochemical studies suggesting that decidual PRL is not stored in secretory granules. The differences in the subcellular localization of PRL in decidual and pituitary tissues may explain, in part, the differences in the regulation of PRL release from these tissues.

An evaluation under code of new techniques for the detection of cytomegalovirus antibodies: sensitivity of assays and importance of immune complexes.

Given the morbidity and occasional mortality associated with cytomegalovirus infection and the requirement for good seroepidemiologic tools, assays of high sensitivity and reliability are needed for detection of cytomegalovirus-specific antibody. We report the successful application of two sensitive techniques, antibody-dependent cellular cytotoxicity (ADCC) and the enzyme-linked immunosorbent assay (ELISA) for the measurement of these antibodies and document their favorable comparison with conventional techniques, complement fixation (CF) and indirect hemagglutination (IHA). A coded panel of 48 sera from patients with culture-proven cytomegalovirus infection and disease controls was tested in a controlled fashion by all four procedures. Among 18 CF-positive sera, specific antibody was detected by IHA, ADCC and ELISA in dilutions 100 to 1000-fold higher than measurable by the CF test. Ten of 15 sera, seronegative by CF, were nevertheless found to be cytomegalovirus antibody-positive when assessed by the other procedures. Further, the importance of circulating antigen-antibody complexes upon ADCC test results was documented. Eight sera, known to be antibody-positive by other techniques but with an unexpectedly low (or negligible) capacity to induced ADCC, were found to be immune complex-positive by the Raji cell assay. Ultracentrifugation, useful in precipitating antigen-antibody complexes, enhanced ADCC activity in five of six sera known to be complex-positive. It is suggested that the simultaneous application of multiple antibody detection systems, particularly the newly-developed ones (ADCC, ELISA) will provide both earlier diagnostic information as well as a more comprehensive understanding of the human host's immune response to infection with cytomegalovirus.