Phosphorylation of G protein-coupled receptors (GPCRs) is a key event for cell signaling and regulation of receptor function. Previously, using tandem mass spectrometry, we identified two ...phosphorylation sites at the distal C-terminal tail of the chemokine receptor CXCR4, but were unable to determine which specific residues were phosphorylated. Here, we demonstrate that serines (Ser) 346 and/or 347 (Ser-346/7) of CXCR4 are phosphorylated upon stimulation with the agonist CXCL12 as well as a CXCR4 pepducin, ATI-2341. ATI-2341, a G
heterotrimer-biased CXCR4 agonist, induced more robust phosphorylation of Ser-346/7 compared with CXCL12. Knockdown of G protein-coupled receptor kinase (GRK) 2, GRK3, or GRK6 reduced CXCL12-induced phosphorylation of Ser-346/7 with GRK3 knockdown having the strongest effect, while inhibition of the conventional protein kinase C (PKC) isoforms, particularly PKC
, reduced phosphorylation of Ser-346/7 induced by either CXCL12 or ATI-2341. The loss of GRK3- or PKC-mediated phosphorylation of Ser-346/7 impaired the recruitment of
-arrestin to CXCR4. We also found that a pseudo-substrate peptide inhibitor for PKC
effectively inhibited CXCR4 phosphorylation and signaling, most likely by functioning as a nonspecific CXCR4 antagonist. Together, these studies demonstrate the role Ser-346/7 plays in arrestin recruitment and initiation of receptor desensitization and provide insight into the dysregulation of CXCR4 observed in patients with various forms of WHIM syndrome.
Binocular vision requires the segregation of retinal ganglion cell (RGC) axons extending from the retina into the ipsilateral and contralateral optic tracts. RGC axon segregation occurs at the optic ...chiasm, which forms at the ventral diencephalon midline. Using expression analyses, retinal explants and genetically modified mice, we demonstrate that CXCL12 (SDF1) is required for axon segregation at the optic chiasm. CXCL12 is expressed by the meninges bordering the optic pathway, and CXCR4 by both ipsilaterally and contralaterally projecting RGCs. CXCL12 or ventral diencephalon meninges potently promoted axon outgrowth from both ipsilaterally and contralaterally projecting RGCs. Further, a higher proportion of axons projected ipsilaterally in mice lacking CXCL12 or its receptor CXCR4 compared with wild-type mice as a result of misrouting of presumptive contralaterally specified RGC axons. Although RGCs also expressed the alternative CXCL12 receptor ACKR3, the optic chiasm developed normally in mice lacking ACKR3. Our data support a model whereby meningeal-derived CXCL12 helps drive axon growth from CXCR4-expressing RGCs towards the diencephalon midline, enabling contralateral axon growth. These findings further our understanding of the molecular and cellular mechanisms controlling optic pathway development.
The chemokine Cxcl12 binds Cxcr4 and Cxcr7 receptors to control cell migration in multiple biological contexts, including brain development, leukocyte trafficking, and tumorigenesis. Both receptors ...are expressed in the CNS, but how they cooperate during migration has not been elucidated. Here, we used the migration of cortical interneurons as a model to study this process. We found that Cxcr4 and Cxcr7 are coexpressed in migrating interneurons, and that Cxcr7 is essential for chemokine signaling. Intriguingly, this process does not exclusively involve Cxcr7, but most critically the modulation of Cxcr4 function. Thus, Cxcr7 is necessary to regulate Cxcr4 protein levels, thereby adapting chemokine responsiveness in migrating cells. This demonstrates that a chemokine receptor modulates the function of another chemokine receptor by controlling the amount of protein that is made available for signaling at the cell surface.
► Cxcr4 and Cxcr7 receptors are coexpressed by migrating neurons ► Cxcr7 function is required for chemotaxis in response to Cxcl12 ► Cxcr7 operates as a sensor for Cxcl12 and adjusts Cxcr4 protein levels accordingly ► Migrating neurons collectively regulate chemokine responsiveness
•CXCL14 modulation of CXCL12-operated CXCR4 regulation and function was examined.•CXCL14 had no effect on CXCL12-induced CXCR4 phosphorylation at serines 346,347.•CXCL12-induced dynamic mass ...redistribution was not changed by CXCL14.•G protein and Erk1,2 signaling of CXCR4 were also unaffected.•CXCL12 should still be considered the only relevant chemokine ligand of CXCR4.
C-X-C motif chemokine 12/C-X-C chemokine receptor type 4 (CXCL12/CXCR4) signaling is involved in ontogenesis, hematopoiesis, immune function and cancer. Recently, the orphan chemokine CXCL14 was reported to inhibit CXCL12-induced chemotaxis – probably by allosteric modulation of CXCR4. We thus examined the effects of CXCL14 on CXCR4 regulation and function using CXCR4-transfected human embryonic kidney (HEK293) cells and Jurkat T cells. CXCL14 did not affect dose–response profiles of CXCL12-induced CXCR4 phosphorylation, G protein-mediated calcium mobilization, dynamic mass redistribution, kinetics of extracellular signal-regulated kinase 1 (ERK1) and ERK2 phosphorylation or CXCR4 internalization. Hence, essential CXCL12-operated functions of CXCR4 are insensitive to CXCL14, suggesting that interactions of CXCL12 and CXCL14 pathways depend on a yet to be identified CXCL14 receptor.
Objective: The overexpression of somatostatin receptor 2 (sst2) in neuroendocrine tumors is the molecular basis for diagnostic and therapeutic application of the stable somatostatin analog ...octreotide. Recent evidence has shown that the immunocytochemical evaluation of sst2A status is of value for predicting response to octreotide therapy and disease prognosis. However, due to the lack of monoclonal and limited availability of specific polyclonal anti-sst2A antibodies, only very few patients can currently benefit from in vitro sst2 evaluation.
Methods: In the present study, we extensively characterized the novel rabbit monoclonal anti-sst2A antibody (clone UMB-1) using tissues from sst2-deficient mice and their wild-type littermates. UMB-1 was then subjected to a comparative study of immunohistochemistry on a series of histological specimens from formalin-fixed, paraffin-embedded human tumors and adjacent normal tissues.
Results: Immunoprecipitation experiments unequivocally demonstrated that UMB-1 selectively detected its cognate sst2A and did not cross-react with other proteins present in crude tissue homogenates. The UMB-1 monoclonal antibody, when compared with currently available polyclonal antisera, yielded several times more effective immunohistochemical staining of fixed-embedded tissues with a predominance of plasma membrane staining and very low cytoplasmic signal even without heat-based antigen retrieval. In addition, dual immunofluorescence revealed for the first time that the sst2A is present on not only gastrin-containing but also ghrelin-containing cells in human gastric mucosa.
Conclusion: Thus, the rabbit monoclonal antibody UMB-1 may prove of great value in the assessment of sst2A status in human neuroendocrine tumors during routine histopathological examination.
Abstract Expression patterns of the second SDF-1 receptor RDC1/CXCR7 were examined after focal ischemia in rats using in situ hybridization. CXCR7 mRNA was identified in the ventricle walls as well ...as neuronal, astroglial, and vascular cells. After ischemia, intact cortical regions showed a rapid, 4 days-lasting increase in neuronal CXCR7 expression. In the ischemic tissue CXCR7 expression was scarce and associated with blood vessels. Between days 2 and 10 after ischemia-onset, SDF-1 expression increased strongly in the peri-infarct and infarct region, which was accompanied by the appearance of numerous CXCR4-expressing but not CXCR7-expressing cells. These patterns suggest that SDF-1 may influence vascular, astroglial, and neuronal functions via CXCR7 and mediate cell recruitment to ischemic brain areas via CXCR4.
Glutamatergic principal neurons, GABAergic interneurons and thalamocortical axons (TCAs) are essential elements of the cerebrocortical network. Principal neurons originate locally from radial glia ...and intermediate progenitors (IPCs), whereas interneurons and TCAs are of extrinsic origin. Little is known how the assembly of these elements is coordinated. C-X-C motif chemokine 12 (CXCL12), which is known to guide axons outside the neural tube and interneurons in the cortex, is expressed in the meninges and IPCs. Using mouse genetics, we dissected the influence of IPC-derived CXCL12 on TCAs and interneurons by showing that Cxcl12 ablation in IPCs, leaving meningeal Cxcl12 intact, attenuates intracortical TCA growth and disrupts tangential interneuron migration in the subventricular zone. In accordance with strong CXCR4 expression in the forming thalamus and TCAs, we identified a CXCR4-dependent growth-promoting effect of CXCL12 on TCAs in thalamus explants. Together, our findings indicate a cell-autonomous role of CXCR4 in promoting TCA growth. We propose that CXCL12 signals from IPCs link cortical neurogenesis to the progression of TCAs and interneurons spatially and temporally. Significance statement: The cerebral cortex exerts higher brain functions including perceptual and emotional processing. Evolutionary expansion of the mammalian cortex is mediated by intermediate progenitors, transient amplifying cells generating cortical excitatory neurons. During the peak period of cortical neurogenesis, migrating precursors of inhibitory interneurons originating in subcortical areas and thalamic axons invade the cortex. Although defects in the assembly of cortical network elements cause neurological and mental disorders, little is known how neurogenesis, interneuron recruitment, and axonal ingrowth are coordinated. We demonstrate that intermediate progenitors release the chemotactic cytokine CXCL12 to promote intracortical interneuron migration and growth of thalamic axons via the cognate receptor CXCR4. This paracrine signal may ensure thalamocortical connectivity and dispersion of inhibitory neurons in the rapidly growing cortex.
Pasireotide (SOM230) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, Cushing's disease, and carcinoid tumors. Whereas octreotide acts ...primarily via the sst(2A) somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst(2A) activity combined with enhanced binding to other somatostatin receptor subtypes. In the present study, we used phophosite-specific antibodies to examine agonist-induced phosphorylation of the rat sst(2A) receptor. We show that somatostatin and octreotide stimulate the complete phosphorylation of a cluster of four threonine residues within the cytoplasmic (353)TTETQRT(359) motif in a variety of cultured cell lines in vitro as well as in intact animals in vivo. This phosphorylation was mediated by G protein-coupled receptor kinases (GRK) 2 and 3 and followed by rapid cointernalization of the receptor and ss-arrestin into the same endocytic vesicles. In contrast, pasireotide failed to promote substantial phosphorylation and internalization of the rat sst(2A) receptor. In the presence of octreotide or SS-14, SOM230 showed partial agonist behavior, inhibiting phosphorylation, and internalization of sst(2A). Upon overexpression of GRK2 or GRK3, pasireotide stimulated selective phosphorylation of Thr356 and Thr359 but not of Thr353 or Thr354 within the (353)TTETQRT(359) motif. Pasireotide-mediated phosphorylation led to the formation of relatively unstable beta-arrestin-sst(2A) complexes that dissociated at or near the plasma membrane. Thus, octreotide and pasireotide are equally active in inducing classical G protein-dependent signaling via the sst(2A) somatostatin receptor. Yet, we find that they promote strikingly different patterns of sst(2A) receptor phosphorylation and, hence, stimulate functionally distinct pools of beta-arrestin.
Adult hippocampal neurogenesis is implicated in learning and memory processing. It is tightly controlled at several levels including progenitor proliferation as well as migration, differentiation and ...integration of new neurons. Hippocampal progenitors and immature neurons reside in the subgranular zone (SGZ) and are equipped with the CXCL12‐receptor CXCR4 which contributes to defining the SGZ as neurogenic niche. The atypical CXCL12‐receptor CXCR7 functions primarily by sequestering extracellular CXCL12 but whether CXCR7 is involved in adult neurogenesis has not been assessed. We report that granule neurons (GN) upregulate CXCL12 and CXCR7 during dentate gyrus maturation in the second postnatal week. To test whether GN‐derived CXCL12 regulates neurogenesis and if neuronal CXCR7 receptors influence this process, we conditionally deleted Cxcl12 and Cxcr7 from the granule cell layer. Cxcl12 deletion resulted in lower numbers, increased dispersion and abnormal dendritic growth of immature GN and reduced neurogenesis. Cxcr7 ablation caused an increase in progenitor proliferation and progenitor numbers and reduced dispersion of immature GN. Thus, we provide a new mechanism where CXCL12‐signals from GN prevent dispersion and support maturation of newborn GN. CXCR7 receptors of GN modulate the CXCL12‐mediated feedback from GN to the neurogenic niche.
Main Points
Mature dentate gyrus granule neurons express CXCL12 and CXCR7.
Neuron‐derived CXCL12 restricts cell dispersion, regulates differentiation and promotes integration of new neurons.
Neuronal CXCR7 influences progenitor behavior and cell dispersion.
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