Role of G protein-coupled receptor kinases in cell migration

G protein-coupled receptor kinases (GRKs) are emerging as important integrative nodes in cell migration processes. Recent evidence links GRKs (particularly the GRK2 isoform) to the complex modulation of diverse aspects of cell motility. In addition to its well-established role in the desensitization of G protein-coupled receptors involved in chemotaxis, GRK2 can play a effector role in the organization of actin and microtubule networks and in adhesion dynamics, by means of novel substrates and transient interacting partners, such as the GIT-1 scaffold or the cytoplasmic α-tubulin deacetylase histone deacetylase 6 (HDAC6). The overall effect of altering GRK levels or activity on chemotaxis would depend on how such different roles are integrated in a given cell type and physiological context, and may have relevant implications in inflammatory diseases or cancer progressionOur laboratory is funded by grants from Ministerio de Educación y Ciencia (SAF2011-23800), Fundación Ramón Areces, The Cardiovascular Network (RECAVA) of Ministerio Sanidad y Consumo-Instituto Carlos III (RD12/0042/0012), Comunidad de Madrid Indisnet Network (S2011/BMD-2332) to F.M, and Fundación Eugenio Rodriguez Pascual, Fundación Ramón Areces and Instituto de Salud Carlos III (PI11/00859) to P.P

The interplay between G protein-coupled receptor kinase 2 (GRK2) and histone deacetylase 6 (HDAC6) at the crossroads of epithelial cell motility

G protein-coupled receptor kinase 2 (GRK2) is emerging as a key integrative node in cell migration control. In addition to its canonical role in the desensitization of G protein-coupled receptors involved in chemotaxis, novel recently identified GRK2 substrates and interacting partners appear to mediate the GRK2-dependent modulation of diverse molecular processes involved in motility, such as gradient sensing, cell polarity or cytoskeletal reorganization. We have recently identified an interaction between GRK2 and histone deacetylase 6 (HDAC6), a major cytoplasmic α-tubulin deacetylase involved in cell motility and adhesion. GRK2 dynamically associates with and phosphorylates HDAC6 to stimulate its α-tubulin deacetylase activity at specific cellular localizations such as the leading edge of migrating cells, thus promoting local tubulin deacetylation and enhanced motility. This GRK2-HDAC6 functional interaction may have important implications in pathological contexts related to aberrant epithelial cell migration

G protein-coupled receptor kinase 2 (GRK2) as a multifunctional signaling hub

Accumulating evidence indicates that G protein-coupled receptor kinase 2 (GRK2) is a versatile protein that acts as a signaling hub by modulating G protein-coupled receptor (GPCR) signaling and also via phosphorylation or scaffolding interactions with an extensive number of non-GPCR cellular partners. GRK2 multifunctionality arises from its multidomain structure and from complex mechanisms of regulation of its expression levels, activity, and localization within the cell, what allows the precise spatio-temporal shaping of GRK2 targets. A better understanding of the GRK2 interactome and its modulation mechanisms is helping to identify the GRK2-interacting proteins and its substrates involved in the participation of this kinase in different cellular processes and pathophysiological contexts.Our laboratories are supported by Ministerio de Ciencia, Innovación y Universidades (Grant SAF2017-84125-R to FM and SAF2017-82886-R to FSM), CIBERCV-Instituto de Salud Carlos III, Spain (Grant B16/11/00278 to F.M, co-funded with European FEDER contribution), Instituto de Salud Carlos III, Spain (Grant PI17-00576 to PP and grant PI18/01662 to CR), Fundacion Ramón Areces (to FM) and Programa de Actividades en Biomedicina de la Comunidad de Madrid-B2017/BMD-3671-INFLAMUNE to FM and FSM. We also acknowledge institutional support to the CBMSO from Fundación Ramón ArecesS

A novel GRK2/HDAC6 interaction modulates cell spreading and motility

Cell motility and adhesion involves dynamic microtubule (MT) acetylation/deacetylation, a process regulated by enzymes as HDAC6, a major cytoplasmic α-tubulin deacetylase. We identify G protein-coupled receptor kinase 2 (GRK2) as a key novel stimulator of HDAC6. GRK2, which levels inversely correlate with the extent of α-tubulin acetylation in epithelial cells and fibroblasts, directly associates with and phosphorylates HDAC6 to stimulate α-tubulin deacetylase activity. Remarkably, phosphorylation of GRK2 itself at S670 specifically potentiates its ability to regulate HDAC6. GRK2 and HDAC6 colocalize in the lamellipodia of migrating cells, leading to local tubulin deacetylation and enhanced motility. Consistently, cells expressing GRK2-K220R or GRK2-S670A mutants, unable to phosphorylate HDAC6, exhibit highly acetylated cortical MTs and display impaired migration and protrusive activity. Finally, we find that a balanced, GRK2/HDAC6-mediated regulation of tubulin acetylation differentially modulates the early and late stages of cellular spreading. This novel GRK2/HDAC6 functional interaction may have important implications in pathological contexts

Crosstalk between CXCR4/ACKR3 and EGFR signaling in breast cancer cells

A better understanding of the complex crosstalk among key receptors and signaling pathways involved in cancer progression is needed to improve current therapies. We have investigated in cell models representative of the major subtypes of breast cancer (BC) the interplay between the chemokine CXCL12/CXCR4/ACKR3 and EGF receptor (EGFR) family signaling cascades. These cell lines display a high heterogeneity in expression profiles of CXCR4/ACKR3 chemokine receptors, with a predominant intracellular localization and different proportions of cell surface CXCR4+, ACKR3+ or double-positive cell subpopulations, and display an overall modest activation of oncogenic pathways in response to exogenous CXCL12 alone. Interestingly, we find that in MDA-MB-361 (luminal B subtype, Her2-overexpressing), but not in MCF7 (luminal A) or MDA-MB-231 (triple negative) cells, CXCR4/ACKR3 and EGFR receptor families share signaling components and crosstalk mechanisms to concurrently promote ERK1/2 activation, with a key involvement of the G protein-coupled receptor kinase 2 (GRK2) signaling hub and the cytosolic tyrosine kinase Src. Our findings suggest that in certain BC subtypes, a relevant cooperation between CXCR4/ACKR3 and growth factor receptors takes place to integrate concurrent signals emanating from the tumor microenvironment and foster cancer progressio

The stress connection in cancer: the adrenergic fuelling of breast tumors

Cancer progression involves complex interactions between tumor cells and the surrounding microenvironment. Chronic psychosocial stress and sympathetic nervous system activation lead to abnormal catecholamine release, impacting tumor cells directly and indirectly and fuelling cancer-promoting effects. However, the same adrenergic Receptor (AR) that mediate these effects could also convey exercise-related beneficial changes. Epidemiological studies show conflicting associations between stress, AR inhibitors, and breast cancer (BC) metastatic progression. Adrenergic sympathetic stress triggers sustained inflammatory and hypoxic-related signaling pathways, alters function and distribution of immune cell populations, and remodels blood vessels, leading to immunosuppression and premetastatic site formation. Activated AR initiate feedback loops with tyrosine kinase receptors and chemokine receptors, affecting stem-related transcription factors, pro-inflammatory mediators, angiogenic factors, and energy metabolism regulators, promoting tumor growth and invasion. Understanding molecular mechanisms of agonistic and antagonistic AR ligands and crosstalk with other signaling pathways is crucial for developing effective therapies targeting adrenergic-driven BC progressionP2022/BMD-7209/INTEGRAMUNE-CM, SEV2016-064

Degradation of GRK2 and AKT is an early and detrimental event in myocardial ischemia/reperfusion

GRK2; AKT; Ischemia-reperfusionGRK2; AKT; Isquemia-reperfusiónGRK2; AKT; Isquèmia-reperfusióBackground: Identification of signaling pathways altered at early stages after cardiac ischemia/reperfusion (I/R) is crucial to develop timely therapies aimed at reducing I/R injury. The expression of G protein-coupled receptor kinase 2 (GRK2), a key signaling hub, is up-regulated in the long-term in patients and in experimental models of heart failure. However, whether GRK2 levels change at early time points following myocardial I/R and its functional impact during this period remain to be established. Methods: We have investigated the temporal changes of GRK2 expression and their potential relationships with the cardioprotective AKT pathway in isolated rat hearts and porcine preclinical models of I/R. Findings: Contrary to the maladaptive up-regulation of GRK2 reported at later times after myocardial infarction, successive GRK2 phosphorylation at specific sites during ischemia and early reperfusion elicits GRK2 degradation by the proteasome and calpains, respectively, thus keeping GRK2 levels low during early I/R in rat hearts. Concurrently, I/R promotes decay of the prolyl-isomerase Pin1, a positive regulator of AKT stability, and a marked loss of total AKT protein, resulting in an overall decreased activity of this pro-survival pathway. A similar pattern of concomitant down-modulation of GRK2/AKT/Pin1 protein levels in early I/R was observed in pig hearts. Calpain and proteasome inhibition prevents GRK2/Pin1/AKT degradation, restores bulk AKT pathway activity and attenuates myocardial I/R injury in isolated rat hearts. Interpretation: Preventing transient degradation of GRK2 and AKT during early I/R might improve the potential of endogenous cardioprotection mechanisms and of conditioning strategies.Our laboratories are supported by Instituto de Salud Carlos III, Spain (grant PI-16/00232; RETICS-RIC-RD12/0042/0021 to DGD, co-funded with European Regional Development Fund-FEDER contribution, and grants PI14-00435 and PI17-00576 to PP), by Ministerio de Economía; Industria y Competitividad (MINECO) of Spain (grant SAF2017-84125-R to F.M.); by CIBERCV-Instituto de Salud Carlos III, Spain (grant CB16/11/00479 to DGD and CB16/11/00278 to F.M, co-funded with European Regional Development Fund-FEDER contribution), and Programa de Actividades en Biomedicina de la Comunidad de Madrid-B2017/BMD-3671-INFLAMUNE to F.M. We also acknowledge institutional support to the CBMSO from Fundación Ramón Areces. This work is dedicated to the memory of our colleague and friend Dr. David García-Dorado, who sadly passed away during the final revision stage of this manuscript

GRK2-Dependent HuR Phosphorylation Regulates HIF1α Activation under Hypoxia or Adrenergic Stress.

Adaptation to hypoxia is a common feature in solid tumors orchestrated by oxygen-dependent and independent upregulation of the hypoxia-inducible factor-1α (HIF-1α). We unveiled that G protein-coupled receptor kinase (GRK2), known to be overexpressed in certain tumors, fosters this hypoxic pathway via phosphorylation of the mRNA-binding protein HuR, a central HIF-1α modulator. GRK2-mediated HuR phosphorylation increases the total levels and cytoplasmic shuttling of HuR in response to hypoxia, and GRK2-phosphodefective HuR mutants show defective cytosolic accumulation and lower binding to HIF-1α mRNA in hypoxic Hela cells. Interestingly, enhanced GRK2 and HuR expression correlate in luminal breast cancer patients. GRK2 also promotes the HuR/HIF-1α axis and VEGF-C accumulation in normoxic MCF7 breast luminal cancer cells and is required for the induction of HuR/HIF1-α in response to adrenergic stress. Our results point to a relevant role of the GRK2/HuR/HIF-1α module in the adaptation of malignant cells to tumor microenvironment-related stresses.This research was funded by the Instituto de Salud Carlos III: PI17-00576; Ministerio de Economia, Industria y Competitividad, Gobierno de Espana: SAF2017-84125-R; Ministerio de Economia, Industria y Competitividad, Gobierno de Espana: SAF2017-87301-R; Instituto de Salud Carlos III: CIBERCV CB16/11/00278; Instituto de Salud Carlos III: PI14-00435; Fundacion Ramon Areces and the Comunidad de Madrid: B2017/BMD-3671-INFLAMUNE.S

Cell-Type Specific GRK2 Interactomes: Pathophysiological Implications

G protein-coupled receptor kinase 2 (GRK2) is emerging as a key hub in cell signaling cascades. In addition to modulating activated G protein-coupled receptors, GRK2 can phosphorylate and/or functionally interact with a complex network of cellular proteins in a cell-type and physiological context-dependent way. A combination of such canonical and noncanonical interactions underlies the participation of this kinase in the control of cell migration, proliferation or metabolism and in integrated processes at the tissue or whole organism levels, such as angiogenesis, cardiovascular function, or insulin resistance, among others. Its role as a signaling node and the fact that altered levels of GRK2 are detected in a variety of pathological conditions put forward this protein as a potentially relevant diagnostic and therapeutic targe