G protein-coupled receptor kinase 2 negatively regulates chemokine signaling at a level downstream from G protein subunits.

The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes ligand-activated G protein-coupled-receptors. Here, evidence is shown for a novel role of GRK2 in regulating chemokine-mediated signals. The presence of increased levels of GRK2 in human embryonic kidney (HEK) 293 cells produced a significant reduction of the extracellular signal-regulated kinase (ERK) response to CCL2. This effect is independent of its role in receptor phosphorylation because the kinase-deficient mutant GRK2K220R was able to reduce this response, and ERK activation by CCR2BIX, a phosphorylation-defective receptor mutant, was also inhibited by GRK2. Constructs containing the Galpha(q)-binding RGS-like RH domain of GRK2 or its Gbetagamma-binding domain could not reproduce the inhibition, thus revealing that GRK2 acts downstream of G proteins. Interestingly, chemokine-driven mitogen-activated protein kinase kinase (MEK) stimulation is not affected in cells overexpressing GRK2 or GRK2K220R or in splenocytes from heterozygous GRK2 mice, where reduced kinase levels correlate with enhanced ERK activation by chemokines. We find GRK2 and MEK in the same multimolecular complex, thus suggesting a mechanism for GRK2 regulation of ERK activity that involves a direct or coordinate interaction with MEK. These results suggest an important role for GRK2 in the control of chemokine induction of ERK activation at the level of the MEK-ERK interface.

Signaling pathways for monocyte chemoattractant protein 1-mediated extracellular signal-regulated kinase activation.

G protein-coupled receptors (GPCRs) initiate diverse down-stream signaling events in response to ligand stimulation, as rapid activation of the extracellular signal-regulated kinase ERK1 and ERK2. The chemokine monocyte chemoattractant protein-1 (MCP-1) is the agonist for several chemokine receptors that belong to the GPCR superfamily, CCR2 being the most important. Stimulation of mitogen-activated protein kinases (MAPKs) by MCP-1 has been implicated in integrin activation and chemotaxis, but the molecular pathways down-stream of the receptors remain unclear. To dissect the cascade of events leading to MAPK activation upon CCR2 receptor stimulation, several specific inhibitors and mutants of signal transduction proteins were used in monocytic cells endogenously expressing CCR2 and/or in human embryonic kidney-293 cells transfected with CCR2B receptors and epitope-tagged ERK1. We show that ERK activation by MCP-1 involves heterotrimeric Gi protein subunits, protein kinase C, phosphoinositide-3-kinase, and Ras. On the other hand, the activity of cytosolic tyrosine kinases, epidermal growth factor receptor transactivation, or variations in intracellular calcium levels are not required for the mitogenic activation elicited by MCP-1. In addition, we find that internalization of CCR2B itself is not necessary for efficient MCP-1-induced activation of ERK, although a dynamin mutant partially inhibits ERK stimulation. These results suggest that different parallel pathways are being activated that lead to the full activation of the mitogen-activated protein kinase cascade and that internalization of other signaling proteins but not of the receptor is required for complete ERK activation.

G protein-coupled receptor kinase 2 (GRK2): mechanisms of regulation and physiological functions.

G protein-coupled receptor kinase 2 (GRK2) plays a key role in determining the rate and extent of G protein-coupled receptor (GPCR) desensitization and resensitization. Recent data indicate that GRK2 activity, subcellular distribution and expression are tightly regulated. The important physiological function of GRK2 as a modulator of the efficacy of GPCR signal transduction systems is exemplified by its relevance in cardiovascular physiopathology as well as by its emerging role in the regulation of chemokine receptors.

Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2.

Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine cytokine family, whose physiological function is mediated by binding to the CCR2 and CCR4 receptors, which are members of the G protein-coupled receptor family. MCP-1 plays a critical role in both activation and migration of leukocytes. Rapid chemokine receptor desensitization is very likely essential for accurate chemotaxis. In this report, we show that MCP-1 binding to the CCR2 receptor in Mono Mac 1 cells promotes the rapid desensitization of MCP-1-induced calcium flux responses. This desensitization correlates with the Ser/Thr phosphorylation of the receptor and with the transient translocation of the G protein-coupled receptor kinase 2 (GRK2, also called beta-adrenergic kinase 1 or betaARK1) to the membrane. We also demonstrate that GRK2 and the uncoupling protein beta-arrestin associate with the receptor, forming a macromolecular complex shortly after MCP-1 binding. Calcium flux responses to MCP-1 in HEK293 cells expressing the CCR2B receptor were also markedly reduced upon cotransfection with GRK2 or the homologous kinase GRK3. Nevertheless, expression of the GRK2 dominant-negative mutant betaARK-K220R did not affect the initial calcium response, but favored receptor response to a subsequent challenge by agonists. The modulation of the CCR2B receptor by GRK2 suggests an important role for this kinase in the regulation of monocyte and lymphocyte response to chemokines.

Centric fission, centromere-telomere fusion and isochromosome formation: a possible origin of a de novo 12p trisomy.

A 5-month-old girl had a typical 12p trisomy syndrome due to a monocentric i(12p) present in a 46-chromosome complement that also included the translocation of all 12q onto the 8p telomere; i.e., her complex karyotype could be written as 46,XX,-8,-12, +der(8),t(8;12)(p23.3;cen),+i(12p). The present concurrence of a whole-arm q translocation and an i(p) for a single chromosome, along with six previous similar instances involving chromosomes 4, 5 and 9, suggests the following origin for such a special rearrangement: a centric fission in G1 initially yielding two telocentrics; at the next replication, the tel(q) translocates onto a nonhomologous telomere (centromere-telomere fusion), whereas the tel(p) becomes an i(p). This mechanism can be either meiotic or postzygotic and surmises that the translocated long arm retains a partial centromere, which subsequently is inactivated and loses its staining properties.

Whole-arm t(X;17) (Xp17q;Xq17p) and gonadal dysgenesis. A further exception to the critical region hypothesis.

A 19-year-old female patient with gonadal dysgenesis and a de novo t(X;17) (Xp17q;Xq17p) is described. Since the critical segment Xq13----q26 was intact, this case is a further exception to the critical region hypothesis.

Aberraciones cromosómicas en 334 individuos con perdidas gestacionales diversas: (incluyendo 144 parejas) / Chromosome aberrations in 334 subjects with repeated abortion (including 144 comples)

La evaluación citogenética de 334 individuos (incluyendo 144 parejas) con abortos repetidos y/o pérdidas gestacionales diversas demostró una aberración cromosómica en 15 de los individuos (4.49%) y en 14 de las parejas (9.72%). La comparación de la frecuencia de anormalidades en individuos que no habían procreado hijos normales contra aquélla de los que sí lo habían hecho (5.62% vs. 1.18%) reveló una diferencia estadísticamente significativa (p<0.02). Estos datos apoyan la conveniencia de efectuar estudios citogenéticos parentales en la investigación etiológica de pérdidas gestacionales, particularmente cuando no ha habido descendencia normal. Tanto los resultados obtenidos como el tipo de anormalidades observadas (8 translocaciones recíprocas, 2 translocaciones Robertsonianas, 2 mosaicos para el cromosoma X, 1 inversión pericéntrica, 1 sitio frágil y 1 anillo no identificado en mosaico) fueron similares a los obtenidos por otros autores

Azoospermia and duplication 3qter as distinct consequences of a familial t(X;3) (q26;q13.2).

A t(X;3) (q26;q13.2) was found in three generations of a family. Female carriers exhibited normal reproductive function, whereas all three postpuberal male carriers showed spermatogenesis arrest at meiosis I. Additionally, a 2 3/12-year-old girl had duplication 3qter resulting from an adjacent-1 segregation.

Monosomy 20p due to a de novo del(20)(p12.2). Clinical and radiological delineation of the syndrome.

A 16 year-old boy with monosomy 20p was studied. The clinical and radiological data compared with those from the three previously reported cases, permit the delineation of a distinct syndrome of low birthweight, flat face, low nasal bridge, long philtrum, short neck, small overfolded ears, chest deformity, kyphoscoliosis, congenital heart defect, hypoplastic or absent ribs and rachischisis (butterfly-shaped vertebral bodies). The critical chromosome segment causing this syndrome is tentatively defined as 20p13.

Type and contretype signs in monosomy and trisomy 9p. On a case 46,XY, del (9) (pter yields p12:).

A 15-month-old male with a partial monosomy 9p is reported. The comparative analysis with other cases of 9p monosomy demonstrates a typical phenotype which when compared to that of 9p trisomy, permits the delineation of fifteen "type and contretype" signs.