High affinity interaction with filamin A protects against calcium-sensing receptor degradation

Calcium-sensing receptors (CaR) regulate cell proliferation, differentiation, and apoptosis through the MAPK pathway. MAPK pathway activation requires the cytoskeletal scaffold protein filamin A. Here we examine the interactions of CaR with filamin A in HEK-293 and M2 or A7 melanoma cells to determine how interactions with filamin A facilitate signaling. Filamin A interacts with CaR through two predicted beta-strands from residues 962 to 981; interactions between filamin A and CaR are greatly enhanced by exposure to 5 mM Ca2+. Truncations or deletions (from 972 to 997 or 962 to 981) of the CaR carboxyl terminus eliminate high affinity interactions with filamin A, but CaR-mediated MAPK pathway activation still occurs. CaR-mediated ERK phosphorylation can be localized to a predicted alpha-helix proximal to the membrane, which has been shown to be important for G protein-mediated signaling (residues 868-879). In M2 cells (-filamin A), CaR expression levels are very low; cotransfection of CaR with filamin A increases total cellular CaR and increases plasma membrane localization of CaR, facilitating CaR signaling to the MAPK pathway; similar results were obtained in HEK-293 cells. Interaction with filamin A increases cellular CaR by preventing CaR degradation, thereby facilitating CaR signaling. In addition, filamin A facilitates signaling to the MAPK pathway even by CaR truncations or deletion mutants that cannot engage in high affinity interactions with filamin A, suggesting the targeting of critical signaling proteins to CaR.     

Direct interaction between the actin-binding protein filamin-A and the inwardly rectifying potassium channel,Kir2.1

The role of filamins in actin cross-linking and membrane stabilization is well established, but recently their ability to interact with a variety of transmembrane receptors and signaling proteins has led to speculation of additional roles in scaffolding and signal transduction. Here we report a direct interaction between filamin-A and Kir2.1, an isoform of inwardly rectifying potassium channel expressed in vascular smooth muscle and an important regulator of vascular tone. Yeast two-hybrid screening of a porcine coronary artery cDNA library using the carboxyl terminus of Kir2.1 as bait yielded cDNA encoding a fragment of filamin-A (residues 2481-2647). Interaction between filamin-A and Kir2.1 was confirmed by in vitro overlay assay of membrane-bound Kir2.1 with glutathione S-transferase fusion protein of the isolated filamin clone. Additionally, antibodies directed against Kir2.1 coimmunoprecipitated filamin-A from arterial smooth muscle cell lysates, and immunocytochemical analysis of individual arterial smooth muscle cells showed that Kir2.1 and filamin co-localize in "hotspots" at the cell membrane. Interaction with filamin-A was found to have no effect on Kir2.1 channel behavior but, rather, increased the number of functional channels resident within the membrane. We conclude that filamin-A is potentially an important regulator of Kir2.1 surface expression and location within vascular smooth muscle.     

The calcium-sensing receptor and its interacting proteins

Seven membrane-spanning, or G protein-coupled receptors were originally thought to act through het-erotrimeric G proteins that in turn activate intracellular enzymes or ion channels, creating relatively simple, linear signalling pathways. Although this basic model remains true in that this family does act via a relatively small number of G proteins, these signalling systems are considerably more complex because the receptors interact with or are located near additional proteins that are often unique to a receptor or subset of receptors. These additional proteins give receptors their unique signalling personalities. The extracellular Ca-sensing receptor (CaR) signals via Galpha(i), Galpha(q) and Galpha(12/13), but its effects in vivo demonstrate that the signalling pathways controlled by these subunits are not sufficient to explain all its biologic effects. Additional structural or signalling proteins that interact with the CaR may explain its behaviour more fully. Although the CaR is less well studied in this respect than other receptors, several CaR-interacting proteins such as filamin, a potential scaffolding protein, receptor activity modifying proteins (RAMPs) and potassium channels may contribute to the unique characteristics of the CaR. The CaR also appears to interact with additional proteins common to other G protein-coupled receptors such as arrestins, G protein receptor kinases, protein kinase C, caveolin and proteins in the ubiquitination pathway. These proteins probably represent a few initial members of CaR-based signalling complex. These and other proteins may not all be associated with the CaR in all tissues, but they form the basis for understanding the complete nature of CaR signalling.     

Calcium-sensing receptor-mediated ERK1/2 activation requires Galphai2 coupling and dynamin-independent receptor internalization

The calcium-sensing receptor (CaR) recently has been shown to activate MAP kinase (ERK1/2) in various cell types as well as in heterologous expression systems. In this study we show that the CaR agonist NPS R-467 (1 microm), which does not activate the CaR by itself, robustly activates ERK1/2 in the presence of a low concentration of Ca(2+) (0.5 mm CaCl(2)) in human embryonic kidney (HEK) cells permanently expressing the human CaR (HEK-hCaR). Ca(2+) (4 mm) also activates ERK1/2 but with differing kinetics. CaR-dependent ERK1/2 activation begins to desensitize to 4 mm Ca(2+) after 10 min, whereas there is no desensitization to NPS R-467/CaCl(2) as late as 4 h. Moreover, recovery from desensitization occurs as rapidly as 30 min with 4 mm CaCl(2). Pretreatment of HEK-hCaR cells with concanavalin A (250 microg/ml) to block CaR internalization completely eliminated the NPS R-467/CaCl(2)-mediated ERK1/2 activation but did not block the 2-min time point of 4 mm Ca(2+)-mediated ERK1/2 activation. Neither dominant-negative dynamin (K44A) nor dominant-negative beta-arrestin inhibited ERK1/2 activation by either CaR agonist treatment, suggesting that CaR-elicited ERK1/2 signaling occurs via a dynamin-independent pathway. Pertussis toxin pretreatment partially attenuated the 4 mm Ca(2+)-ERK1/2 activation; this attenuated activity was completely restored by co-expression of the Galpha(i2) (C351I) but not Galpha(i1) (C351I) or Galpha(i3) (C351I) G proteins, PTX-insensitive G protein mutants. Taken together, these data suggest that both 4 mm Ca(2+) and NPS R-467/CaCl(2) activate ERK1/2 via distinguishable pathways in HEK-hCaR cells and may represent a nexus to differentially regulate differentiation versus proliferation via CaR activation.     

Filamin-A binds to the carboxyl-terminal tail of the calcium-sensing receptor, an interaction that participates in CaR-mediated activation of mitogen-activated protein kinase

The G protein-coupled, extracellular calcium-sensing receptor (CaR) regulates parathyroid hormone secretion and parathyroid cellular proliferation as well as the functions of diverse other cell types. The CaR resides in caveolae-plasma membrane microdomains containing receptors and associated signaling molecules that are thought to serve as cellular "message centers." An additional mechanism for coordinating cellular signaling is the presence of scaffold proteins that bind and organize components of signal transduction cascades. With the use of the yeast two-hybrid system, we identified filamin-A (an actin-cross-linking, putative scaffold protein that binds mitogen-activated protein kinase (MAPK) components activated by the CaR) as an intracellular binding partner of the CaR's carboxyl (COOH)-terminal tail. A direct interaction of the two proteins was confirmed by an in vitro binding assay. Moreover, confocal microscopy combined with two color immunofluorescence showed co-localization of the CaR and filamin-A within parathyroid cells as well as HEK-293 cells stably transfected with the CaR. Deletion mapping localized the sites of interaction between the two proteins to a stretch of 60 amino acid residues within the distal portion of the CaR's COOH-terminal tail and domains 14 and 15 in filamin-A, respectively. Finally, introducing the portion of filamin-A interacting with the CaR into CaR-transfected HEK-293 cells using protein transduction with a His-tagged, Tat-filamin-A fusion protein nearly abolished CaR-mediated activation of ERK1/2 MAPK but had no effect on ERK1/2 activity stimulated by ADP. Therefore, the binding of the CaR's COOH-terminal tail to filamin-A may contribute to its localization in caveolae, link it to the actin-based cytoskeleton, and participate in CaR-mediated activation of MAPK.     

Structural interaction and functional regulation of polycystin-2 by filamin

Filamins are important actin cross-linking proteins implicated in scaffolding, membrane stabilization and signal transduction, through interaction with ion channels, receptors and signaling proteins. Here we report the physical and functional interaction between filamins and polycystin-2, a TRP-type cation channel mutated in 10-15% patients with autosomal dominant polycystic kidney disease. Yeast two-hybrid and GST pull-down experiments demonstrated that the C-termini of filamin isoforms A, B and C directly bind to both the intracellular N- and C-termini of polycystin-2. Reciprocal co-immunoprecipitation experiments showed that endogenous polycystin-2 and filamins are in the same complexes in renal epithelial cells and human melanoma A7 cells. We then examined the effect of filamin on polycystin-2 channel function by electrophysiology studies with a lipid bilayer reconstitution system and found that filamin-A substantially inhibits polycystin-2 channel activity. Our study indicates that filamins are important regulators of polycystin-2 channel function, and further links actin cytoskeletal dynamics to the regulation of this channel protein.     

Ca2+-sensing receptor induces Rho kinase-mediated actin stress fiber assembly and altered cell morphology, but not in response to aromatic amino acids

The Ca²⁺-sensing receptor (CaR) is a pleiotropic, type III G protein-coupled receptor (GPCR) that associates functionally with the cytoskeletal protein filamin. To investigate the effect of CaR signaling on the cytoskeleton, human embryonic kidney (HEK)-293 cells stably transfected with CaR (CaR-HEK) were incubated with CaR agonists in serum-free medium for up to 3 h. Addition of the calcimimetic NPS R-467 or exposure to high extracellular Ca²⁺ or Mg²⁺ levels elicited actin stress fiber assembly and process retraction in otherwise stellate cells. These responses were ablated by cotreatment with the calcilytic NPS 89636 and were absent in vector-transfected HEK-293 cells. Cotreatment with the Rho kinase inhibitors Y-27632 and H1152 attenuated the CaR-induced morphological change but not intracellular Ca²⁺ (Ca_i²⁺) mobilization or ERK activation, although transfection with a dominant-negative RhoA-binding protein also inhibited calcimimetic-induced actin stress fiber assembly. CaR effects on morphology were unaffected by inhibition of G_q/11 or G_i/o signaling, epidermal growth factor receptor, or the metalloproteinases. In contrast, CaR-induced cytoskeletal changes were not induced by the aromatic amino acids, treatments that also failed to potentiate CaR-induced ERK activation despite inducing Ca_i²⁺ mobilization. Together, these data establish that CaR can elicit Rho-mediated changes in stress fiber assembly and cell morphology, which could contribute to the receptor's physiological actions. In addition, this study provides further evidence that aromatic amino acids elicit differential signaling from other CaR agonists. cytoskeleton; signaling     

Plugging PKA into ERK scaffolds

Cancers often arise in part through derangements in protein kinase signaling. A striking example of this is the finding that approximately 30% of human tumors have mutations in Ras or B-Raf, leading to aberrant ERK kinase activation. Kinase signaling networks are often organized by scaffolding and anchoring proteins that help shape the dynamics of signal processing. AKAP-Lbc associates with the ERK scaffold protein KSR-1 to organize a growth factor and cAMP responsive signaling network. AKAP-Lbc also directs PKA phosphorylation of KSR-1 on a critical residue to ensure maximal signaling efficiency.     

Signaling complexes of the FERM domain-containing protein GRSP1 bound to ARF exchange factor GRP1.

GRP1 is a member of a family of proteins that contain a coiled-coil region, a Sec7 homology domain with guanosine nucleotide exchange activity for the ARF GTP-binding proteins, and a pleckstrin homology domain at the C terminus. The pleckstrin homology domain of GRP1 binds phosphatidylinositol (3,4,5) trisphosphate and mediates the translocation of GRP1 to the plasma membrane upon agonist stimulation of PI 3-kinase activity. Using a (32)P-labeled GRP1 probe to screen a mouse brain cDNA expression library, we isolated a cDNA clone encoding a GRP1-binding partner (GRSP1) that exists as two different splice variants in brain and lung. The GRSP1 protein contains a FERM protein interaction domain as well as two coiled coil domains and may therefore function as a scaffolding protein. Mapping experiments revealed that the interaction of GRP1 and GRSP1 occurs through the coiled coil domains in the two proteins. Immunodepletion experiments indicate that virtually all of the endogenous GRSP1 protein exists as a complex with GRP1 in lung. When co-expressed in Chinese hamster ovary cells expressing the human insulin receptor, both proteins display a diffuse, cytoplasmic localization. Acute translocation and co-localization of GRSP1 and GRP1 to ruffles in the plasma membrane was evident after insulin stimulation. These results identify GRSP1 as a novel member of GRP1 signaling complexes that are acutely recruited to plasma membrane ruffles in response to insulin receptor signaling.     

A new member of the LIM protein family binds to filamin B and localizes at stress fibers

Human filamins are 280-kDa proteins containing an N-terminal actin-binding domain followed by 24 characteristic repeats. They also interact with a number of other cellular proteins. All of those identified to date, with the exception of actin, bind to the C-terminal third of a filamin. In a yeast two-hybrid search of a human placental library, using as bait repeats 10-18 of filamin B, we isolated a cDNA coding for a novel 374 amino acid protein containing a proline-rich domain near its N terminus and two LIM domains at its C terminus. We term this protein filamin-binding LIM protein-1, FBLP-1. Yeast two-hybrid studies with deletion mutants localized the areas of interaction in FBLP-1 to its N-terminal domain and in filamin B to repeats 10-13. FBLP-1 mRNA was detected in a variety of tissues and cells including platelets and endothelial cells. We also have identified two FBLP-1 variants. Both contain three C-terminal LIM domains, but one lacks the N-terminal proline-rich domain. Transfection of FBLP-1 into 293A cells promoted stress fiber formation, and both FBLP-1 and filamin B localized to stress fibers in the transfected cells. The association between filamin B and FBLP-1 may play a hitherto unknown role in cytoskeletal function, cell adhesion, and cell motility.     

GRFbeta, a novel regulator of calcium signaling, is expressed in pancreatic beta cells and brain.

By screening for genes expressed differentially in pancreatic beta cells, we have isolated a cDNA encoding GRFbeta, a novel 178-amino acid protein whose N terminus is identical to that of GRF1, a calcium-dependent guanine nucleotide exchange factor, and whose C terminus is unrelated to known proteins. We show that both GRF1 and GRFbeta are expressed selectively in beta cell lines, pancreatic islet cells and brain. Treatment of beta cell lines (betaTC1 and HIT) with calcium ionophore led to a significant elevation in activity of the Ras signal transduction pathway, as determined by phosphorylation of extracellular signal-related kinase (ERK). Transfection of beta cells with a plasmid encoding a dominant negative variant of GRF1 led to 70% reduction in ERK phosphorylation, consistent with a role for GRF1 in calcium-dependent Ras signaling in these cells. To examine the possible function of GRFbeta, cultured cells were transfected with a GRFbeta expression vector. This led to a significant reduction in both GRF1-dependent ERK phosphorylation and AP1-dependent reporter gene activity. The results suggest that GRF1 plays a role in mediating calcium-dependent signal transduction in beta cells and that GRFbeta represents a novel dominant negative modulator of Ras signaling.     

Human ribosomal protein S3a: cloning of the cDNA and primary structure of the protein.

The amino acid (aa) sequence of human ribosomal protein S3a (hRPS3a) was deduced partially from the nucleotide sequence of the corresponding cDNA and confirmed by direct aa sequencing from the N terminus of the purified hRPS3a protein. The cDNA clone was isolated from a cDNA expression library in the pEX vector using antibodies. The hRPS3a protein has 263 aa and its calculated M(r) is 29 813.     

Tyrosine kinase 2 interacts with and phosphorylates receptor for activated C kinase-1, a WD motif-containing protein

Receptor for activated C kinase (Rack)-1 is a protein kinase C-interacting protein, and contains a WD repeat but has no enzymatic activity. In addition to protein kinase C, Rack-1 also binds to Src, phospholipase Cgamma, and ras-GTPase-activating proteins. Thus, Rack-1 is thought to function as a scaffold protein that recruits specific signaling elements. In a cytokine signaling cascade, Rack-1 has been reported to interact with the IFN-alphabeta receptor and Stat1. In addition, we show here that Rack-1 associates with a member of Jak, tyrosine kinase 2 (Tyk2). Rack-1 interacts weakly with the kinase domain and interacts strongly with the pseudokinase domain of Tyk2. Rack-1 associates with Tyk2 via two regions, one in the N terminus and one in the middle portion (aa 138-203) of Rack-1. Jak activation causes the phosphorylation of tyrosine 194 on Rack-1. After phosphorylation, Rack-1 is translocated toward the perinuclear region. In addition to functioning as a scaffolding protein, these results raise the possibility that Rack-1 functions as a signaling molecule in cytokine signaling cascades.     

Cooperative regulation of extracellular signal-regulated kinase activation and cell shape change by filamin A and beta-arrestins

beta-Arrestins (betaarr) are multifunctional adaptor proteins that can act as scaffolds for G protein-coupled receptor activation of mitogen-activated protein kinases (MAPK). Here, we identify the actin-binding and scaffolding protein filamin A (FLNA) as a betaarr-binding partner using Son of sevenless recruitment system screening, a classical yeast two-hybrid system, coimmunoprecipitation analyses, and direct binding in vitro. In FLNA, the betaarr-binding site involves tandem repeat 22 in the carboxyl terminus. betaarr binds FLNA through both its N- and C-terminal domains, indicating the presence of multiple binding sites. We demonstrate that betaarr and FLNA act cooperatively to activate the MAPK extracellular signal-regulated kinase (ERK) downstream of activated muscarinic M1 (M1MR) and angiotensin II type 1a (AT1AR) receptors and provide experimental evidence indicating that this phenomenon is due to the facilitation of betaarr-ERK2 complex formation by FLNA. In Hep2 cells, stimulation of M1MR or AT1AR results in the colocalization of receptor, betaarr, FLNA, and active ERK in membrane ruffles. Reduction of endogenous levels of betaarr or FLNA and a catalytically inactive dominant negative MEK1, which prevents ERK activation, inhibit membrane ruffle formation, indicating the functional requirement for betaarr, FLNA, and active ERK in this process. Our results indicate that betaarr and FLNA cooperate to regulate ERK activation and actin cytoskeleton reorganization.     

Amino acid-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor are mediated by a phospholipase C/inositol 1,4,5-trisphosphate-independent pathway that requires G12, Rho, filamin-A, and the actin cytoskeleton

The G protein-coupled Ca(2+)-sensing receptor (CaR) is an allosteric protein that responds to two different agonists, Ca(2+) and aromatic amino acids, with the production of sinusoidal or transient oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). Here, we examined whether these differing patterns of [Ca(2+)](i) oscillations produced by the CaR are mediated by separate signal transduction pathways. Using real time imaging of changes in phosphatidylinositol 4,5-biphosphate hydrolysis and generation of inositol 1,4,5-trisphosphate in single cells, we found that stimulation of CaR by an increase in the extracellular Ca(2+) concentration ([Ca(2+)](o)) leads to periodic synthesis of inositol 1,4,5-trisphosphate, whereas l-phenylalanine stimulation of the CaR does not induce any detectable change in the level this second messenger. Furthermore, we identified a novel pathway that mediates transient [Ca(2+)](i) oscillations produced by the CaR in response to l-phenylalanine, which requires the organization of the actin cytoskeleton and involves the small GTPase Rho, heterotrimeric proteins of the G(12) subfamily, the C-terminal region of the CaR, and the scaffolding protein filamin-A. Our model envisages that Ca(2+) or amino acids stabilize unique CaR conformations that favor coupling to different G proteins and subsequent activation of distinct downstream signaling pathways.