scholarly journals Growth Hormone-induced JAK2 Signaling and GH Receptor Down-regulation: Role of GH Receptor Intracellular Domain Tyrosine Residues

Endocrinology ◽  
2012 ◽  
Vol 153 (5) ◽  
pp. 2311-2322 ◽  
Author(s):  
Luqin Deng ◽  
Jing Jiang ◽  
Stuart J. Frank
Keyword(s):  
Tyrosine Phosphorylation ◽  
Metabolic Effects ◽  
Wild Type ◽  
Down Regulation ◽  
Mutant Receptor ◽  
Tyrosine Residues ◽  
Gh Receptor ◽  
Tyrosine Kinase 2 ◽  
Activation Mechanisms ◽  
Type Receptor

GH receptor (GHR) mediates important somatogenic and metabolic effects of GH. A thorough understanding of GH action requires intimate knowledge of GHR activation mechanisms, as well as determinants of GH-induced receptor down-regulation. We previously demonstrated that a GHR mutant in which all intracellular tyrosine residues were changed to phenylalanine was defective in its ability to activate signal transducer and activator of transcription (STAT)5 and deficient in GH-induced down-regulation, but able to allow GH-induced Janus family of tyrosine kinase 2 (JAK2) activation. We now further characterize the signaling and trafficking characteristics of this receptor mutant. We find that the mutant receptor's extracellular domain conformation and its interaction with GH are indistinguishable from the wild-type receptor. Yet the mutant differs greatly from the wild-type in that GH-induced JAK2 activation is augmented and far more persistent in cells bearing the mutant receptor. Notably, unlike STAT5 tyrosine phosphorylation, GH-induced STAT1 tyrosine phosphorylation is retained and augmented in mutant GHR-expressing cells. The defective receptor down-regulation and persistent JAK2 activation of the mutant receptor do not depend on the sustained presence of GH or on the cell's ability to carry out new protein synthesis. Mutant receptors that exhibit resistance to GH-induced down-regulation are enriched in the disulfide-linked form of the receptor, which reflects the receptor's activated conformation. Furthermore, acute GH-induced internalization, a proximal step in down-regulation, is markedly impaired in the mutant receptor compared to the wild-type receptor. These findings are discussed in the context of determinants and mechanisms of regulation of GHR down-regulation.

Deletion of the kinase insert sequence of the platelet-derived growth factor beta-receptor affects receptor kinase activity and signal transduction

1990 ◽  
Vol 10 (2) ◽  
pp. 801-809
Author(s):  
L Severinsson ◽  
B Ek ◽  
K Mellström ◽  
L Claesson-Welsh ◽  
C H Heldin
Keyword(s):  
Growth Factor ◽  
Protein Tyrosine Kinase ◽  
Catalytic Efficiency ◽  
Kinase Domain ◽  
Platelet Derived Growth Factor ◽  
Tyrosine Kinase Domain ◽  
Wild Type ◽  
Mutant Receptor ◽  
Beta Receptor ◽  
Type Receptor

A characteristic feature of the platelet-derived growth factor (PDGF) beta-receptor is the presence of an insert sequence in the protein tyrosine kinase domain. A receptor mutant which lacks the entire insert of 98 amino acids was expressed in CHO cells, and its functional characteristics were compared with those of the wild-type receptor. The mutant receptor bound PDGF-BB with high affinity and mediated internalization and degradation of the ligand with efficiency similar to that of the wild-type receptor but did not transduce a mitogenic signal. It was found to display a decreased autophosphorylation after ligand stimulation and had a decreased ability to phosphorylate exogenous substrates; phosphofructokinase was not phosphorylated at all, whereas a peptide substrate was phosphorylated, albeit at a lower rate compared with phosphorylation by the wild-type receptor. Furthermore, the mutant receptor did not mediate actin reorganization but mediated an increase in c-fos expression. The data indicate that the insert in the kinase domain of the PDGF beta-receptor is important for the substrate specificity or catalytic efficiency of the kinase; the deletion of the insert interferes with the transduction of some, but not all, of the signals that arise after activation of the receptor.

scholarly journals A constitutively active mutant of the α1B-adrenergic receptor can cause greater agonist-dependent down-regulation of the G-proteins Gqα and G11α than the wild-type receptor

1996 ◽  
Vol 320 (1) ◽  
pp. 79-86 ◽  
Author(s):  
Tae Weon LEE ◽  
Alan WISE ◽  
Susanna COTECCHIA ◽  
Graeme MILLIGAN
Keyword(s):  
G Proteins ◽  
Cell Lines ◽  
Adrenergic Receptor ◽  
Inositol Phosphate ◽  
Wild Type ◽  
Down Regulation ◽  
Eta Receptor ◽  
Type Receptor ◽  
In Cells ◽  
Constitutively Active

Rat 1 fibroblasts transfected to express either the wild-type hamster α1B-adrenergic receptor or a constitutively active mutant (CAM) form of this receptor resulting from the alteration of amino acid residues 288–294 to encode the equivalent region of the human β2-adrenergic receptor were examined. The basal level of inositol phosphate generation in cells expressing the CAMα1B-adrenergic receptor was greater than for the wild-type receptor. The addition of maximally effective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the CAMα1B-adrenergic receptor, although this receptor was expressed at lower steady-state levels than the wild-type receptor. The potency of both phenylephrine and noradrenaline to stimulate inositol phosphate production was approx. 200-fold greater at the CAMα1B-adrenergic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ETA receptor, displayed similar potency and maximal effects in the two cell lines. The sustained presence of phenylephrine resulted in down-regulation of the α subunits of the phosphoinositidase C-linked, pertussis toxin-insensitive, G-proteins Gq and G11 in cells expressing either the wild-type or the CAMα1B-adrenergic receptor. The degree of down-regulation achieved was substantially greater in cells expressing the CAMα1B-adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greater potency at the CAMα1B-adrenergic receptor than at the wild-type receptor. There were no detectable differences in the basal rate of Gqα/G11α degradation between cells expressing the wild-type or the CAMα1B-adrenergic receptor. In both cell lines the addition of phenylephrine substantially increased the rate of degradation of these G-proteins, with a greater effect at the CAMα1B-adrenergic receptor. The enhanced capacity of agonist both to stimulate second-messenger production at the CAMα1B-adrenergic receptor and to regulate cellular levels of its associated G-proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to Gq and G11.

PDGF alpha-receptor mediated cellular responses are not dependent on Src family kinases in endothelial cells

1998 ◽  
Vol 111 (5) ◽  
pp. 607-614
Author(s):  
R. Hooshmand-Rad ◽  
K. Yokote ◽  
C.H. Heldin ◽  
L. Claesson-Welsh
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Kinases ◽  
Src Family Kinases ◽  
Wild Type ◽  
Actin Reorganization ◽  
Aortic Endothelial Cells ◽  
Mutant Receptor ◽  
Juxtamembrane Region ◽  
Src Family Tyrosine Kinases ◽  
Type Receptor

Two novel autophosphorylation sites in the juxtamembrane region of the PDGF alpha-receptor, Tyr-572 and Tyr-574, were identified. A Y572/574F mutant PDGF (alpha)-receptor was generated and stably expressed in porcine aortic endothelial cells. In contrast to the wild-type receptor, the mutant receptor was unable to associate with or activate Src family tyrosine kinases. Tyrosine phosphorylated synthetic peptides representing the juxtamembrane sequence of the receptor dose-dependently inhibited the binding of Src family tyrosine kinases to the autophosphorylated PDGF alpha-receptor. The mutant receptor showed similar PDGF-induced kinase activity and ability to mediate mitogenicity, actin reorganization and chemotaxis as the wild-type receptor. Thus activation of Src family kinases by the PDGF alpha-receptor is not essential for PDGF-induced mitogenicity or actin reorganization.

The calcium-dependent activity of large-conductance, calcium-activated K+ channels is enhanced by Pyk2- and Hck-induced tyrosine phosphorylation

2004 ◽  
Vol 287 (3) ◽  
pp. C698-C706 ◽  
Author(s):  
Shizhang Ling ◽  
Jian-Zhong Sheng ◽  
Andrew P. Braun
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Free Calcium ◽  
Wild Type ◽  
Src Family Kinase ◽  
Src Tyrosine Kinase ◽  
Α Subunit ◽  
Calcium Dependent ◽  
Tyrosine Kinase 2 ◽  
Hematopoietic Cell Kinase

Recent results showing that large-conductance, calcium-activated K+ (BKCa) channels undergo direct tyrosine phosphorylation in the presence of c-Src tyrosine kinase have suggested the involvement of these channels in Src-mediated signaling pathways. Given the important role for c-Src in integrin-mediated signal transduction, we have examined the potential regulation of BKCa channels by proline-rich tyrosine kinase 2 (Pyk2), a calcium-sensitive tyrosine kinase activated upon integrin stimulation. Transient coexpression of murine BKCa channels with either wild-type Pyk2 or hematopoietic cell kinase (Hck), a Src-family kinase, led to an enhancement of BKCa channel activity over the range of 1–10 μM free calcium, whereas coexpression with catalytically inactive forms of either kinase did not significantly alter BKCa gating compared with channels expressed alone. In the presence of either wild-type Pyk2 or Hck, BKCa α-subunits were found to undergo tyrosine phosphorylation, as determined by immunoprecipitation and Western blotting strategies. However, tyrosine phosphorylation of the BKCa α-subunit was not detected for channels expressed alone or together with inactive forms of either Pyk2 or Hck. Interestingly, wild-type, but not inactive, Pyk2 was also present in BKCa channel immunoprecipitates, suggesting that Pyk2 may coassociate with the BKCa channel complex after phosphorylation. Collectively, the observed modulation and phosphorylation of BKCa channels by Pyk2 and a Src-family kinase may reflect a general cellular mechanism by which G protein-coupled receptor and/or integrin activation leads to the regulation of membrane ion channels.

scholarly journals Angiotensin II impairs endothelial function via tyrosine phosphorylation of the endothelial nitric oxide synthase

2009 ◽  
Vol 206 (13) ◽  
pp. 2889-2896 ◽  
Author(s):  
Annemarieke E. Loot ◽  
Judith G. Schreiber ◽  
Beate Fisslthaler ◽  
Ingrid Fleming
Keyword(s):  
Angiotensin Ii ◽  
Endothelial Dysfunction ◽  
Tyrosine Phosphorylation ◽  
Dominant Negative ◽  
No Production ◽  
Dependent Manner ◽  
Ang Ii ◽  
Wild Type ◽  
Tyrosine Kinase 2

Proline-rich tyrosine kinase 2 (PYK2) can be activated by angiotensin II (Ang II) and reactive oxygen species. We report that in endothelial cells, Ang II enhances the tyrosine phosphorylation of endothelial NO synthase (eNOS) in an AT1-, H2O2-, and PYK2-dependent manner. Low concentrations (1–100 µmol/liter) of H2O2 stimulated the phosphorylation of eNOS Tyr657 without affecting that of Ser1177, and attenuated basal and agonist-induced NO production. In isolated mouse aortae, 30 µmol/liter H2O2 induced phosphorylation of eNOS on Tyr657 and impaired acetylcholine-induced relaxation. Endothelial overexpression of a dominant-negative PYK2 mutant protected against H2O2-induced endothelial dysfunction. Correspondingly, carotid arteries from eNOS−/− mice overexpressing the nonphosphorylatable eNOS Y657F mutant were also protected against H2O2. In vivo, 3 wk of treatment with Ang II considerably increased levels of Tyr657-phosphorylated eNOS in the aortae of wild-type but not Nox2y/− mice, and this was again associated with a clear impairment in endothelium-dependent vasodilatation in the wild-type but not in the Nox2y/− mice. Collectively, endothelial PYK2 activation by Ang II and H2O2 causes the phosphorylation of eNOS on Tyr657, attenuating NO production and endothelium-dependent vasodilatation. This mechanism may contribute to the endothelial dysfunction observed in cardiovascular diseases associated with increased activity of the renin–angiotensin system and elevated redox stress.

scholarly journals Colony-stimulating factor 1-induced STAT1 and STAT3 activation is accompanied by phosphorylation of Tyk2 in macrophages and Tyk2 and JAK1 in fibroblasts

Blood ◽  
1995 ◽  
Vol 86 (8) ◽  
pp. 2948-2956 ◽  
Author(s):  
U Novak ◽  
AG Harpur ◽  
L Paradiso ◽  
V Kanagasundaram ◽  
A Jaworowski ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factors ◽  
Tyrosine Phosphorylation ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Macrophage Cell Line ◽  
Macrophage Cell ◽  
Stat Proteins ◽  
Type Receptor ◽  
Stimulating Factor

Abstract Colony-stimulating factor 1 (CSF-1) causes the activation of STAT1 and STAT3 transcription factors in bone marrow macrophages (BMM), in the macrophage cell line BAC1.2F5, and in fibroblasts that express the wild-type receptor for CSF-1. Fibroblasts expressing a mutant receptor in which the tyrosine 809 is replaced with phenylalanine do not activate STAT proteins in response to CSF-1. The activation of the STAT proteins in BMM is accompanied by tyrosine phosphorylation of Tyk2. In fibroblasts, the activation of the STAT proteins is accompanied by tyrosine phosphorylation of Tyk2 and JAK1. We propose that these JAK kinases are subjected to very rapid phosphorylation in response to CSF-1, followed by rapid dephosphorylation. Furthermore, we propose that kinases other than JAK kinase may be involved in the phosphorylation of the STAT proteins in response to CSF-1.

scholarly journals Deletion of the kinase insert sequence of the platelet-derived growth factor beta-receptor affects receptor kinase activity and signal transduction.

1990 ◽  
Vol 10 (2) ◽  
pp. 801-809 ◽  
Author(s):  
L Severinsson ◽  
B Ek ◽  
K Mellström ◽  
L Claesson-Welsh ◽  
C H Heldin
Keyword(s):  
Growth Factor ◽  
Protein Tyrosine Kinase ◽  
Catalytic Efficiency ◽  
Kinase Domain ◽  
Platelet Derived Growth Factor ◽  
Tyrosine Kinase Domain ◽  
Wild Type ◽  
Mutant Receptor ◽  
Beta Receptor ◽  
Type Receptor

A characteristic feature of the platelet-derived growth factor (PDGF) beta-receptor is the presence of an insert sequence in the protein tyrosine kinase domain. A receptor mutant which lacks the entire insert of 98 amino acids was expressed in CHO cells, and its functional characteristics were compared with those of the wild-type receptor. The mutant receptor bound PDGF-BB with high affinity and mediated internalization and degradation of the ligand with efficiency similar to that of the wild-type receptor but did not transduce a mitogenic signal. It was found to display a decreased autophosphorylation after ligand stimulation and had a decreased ability to phosphorylate exogenous substrates; phosphofructokinase was not phosphorylated at all, whereas a peptide substrate was phosphorylated, albeit at a lower rate compared with phosphorylation by the wild-type receptor. Furthermore, the mutant receptor did not mediate actin reorganization but mediated an increase in c-fos expression. The data indicate that the insert in the kinase domain of the PDGF beta-receptor is important for the substrate specificity or catalytic efficiency of the kinase; the deletion of the insert interferes with the transduction of some, but not all, of the signals that arise after activation of the receptor.

scholarly journals Signaling Functions of the Tyrosine Residues in the βc Chain of the Granulocyte-Macrophage Colony-Stimulating Factor Receptor

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4759-4766 ◽  
Author(s):  
Keiko Okuda ◽  
Lorie Smith ◽  
James D. Griffin ◽  
Rosemary Foster
Keyword(s):  
Tyrosine Phosphorylation ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Tyrosine Residues ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor ◽  
Stat Pathway

The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) is a heterodimeric receptor expressed by myeloid lineage cells. Binding of GM-CSF activates at least one receptor-associated tyrosine kinase, JAK2, and rapidly induces tyrosine phosphorylation of the GMR βc-chain (GMRβ), but not the GMR α-chain (GMRα). To examine the role of GMRβ tyrosine phosphorylaiton, each of the 8 tyrosine residues in the cytoplasmic domain of the human GMRβ was mutated to phenylalanine (GMRβ-F8), and this mutant receptor was expressed with wild-type GMRα in the interleukin-3–dependent murine hematopoietic cell line, Ba/F3. GM-CSF induced tyrosine phosphorylation of multiple cellular proteins in cells expressing GMRβ-F8 , including JAK2 and STAT5. However, GM-CSF–induced tyrosine phosphorylation of both SHP2 and SHC was reduced or absent compared with wild-type. Next, a series of 8 receptors were generated, each containing only a single, restored, tyrosine residue. Tyrosine 577 was found to be sufficient to regenerate GM-CSF–dependent phosphorylation of SHC, and any of Y577, Y612, or Y695 was sufficient to regenerate GM-CSF–inducible phosphorylation of SHP2. Despite the signaling defect to SHC and SHP2, Ba/F3 cells expressing GMRβ-F8 were still able to proliferate in response to 10 ng/mL of human GM-CSF, although mitogenesis was impaired compared with wild-type GMRβ, and this effect was even more prominent at lower concentrations of GM-CSF (1 ng/mL). Overall, these results indicate that GMRβ tyrosine residues are not necessary for activation of the JAK/STAT pathway or for proliferation, viability, or adhesion signaling in Ba/F3 cells, although tyrosine residues significantly affect the magnitude of the response. However, specific tyrosine residues are needed for activation of SHC and SHP2.

scholarly journals Colony-stimulating factor 1-induced STAT1 and STAT3 activation is accompanied by phosphorylation of Tyk2 in macrophages and Tyk2 and JAK1 in fibroblasts

Blood ◽  
1995 ◽  
Vol 86 (8) ◽  
pp. 2948-2956 ◽  
Author(s):  
U Novak ◽  
AG Harpur ◽  
L Paradiso ◽  
V Kanagasundaram ◽  
A Jaworowski ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factors ◽  
Tyrosine Phosphorylation ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Macrophage Cell Line ◽  
Macrophage Cell ◽  
Stat Proteins ◽  
Type Receptor ◽  
Stimulating Factor

Colony-stimulating factor 1 (CSF-1) causes the activation of STAT1 and STAT3 transcription factors in bone marrow macrophages (BMM), in the macrophage cell line BAC1.2F5, and in fibroblasts that express the wild-type receptor for CSF-1. Fibroblasts expressing a mutant receptor in which the tyrosine 809 is replaced with phenylalanine do not activate STAT proteins in response to CSF-1. The activation of the STAT proteins in BMM is accompanied by tyrosine phosphorylation of Tyk2. In fibroblasts, the activation of the STAT proteins is accompanied by tyrosine phosphorylation of Tyk2 and JAK1. We propose that these JAK kinases are subjected to very rapid phosphorylation in response to CSF-1, followed by rapid dephosphorylation. Furthermore, we propose that kinases other than JAK kinase may be involved in the phosphorylation of the STAT proteins in response to CSF-1.

Functional domains of the very low density lipoprotein receptor: molecular analysis of ligand binding and acid-dependent ligand dissociation mechanisms

1999 ◽  
Vol 112 (19) ◽  
pp. 3269-3281 ◽  
Author(s):  
I. Mikhailenko ◽  
W. Considine ◽  
K.M. Argraves ◽  
D. Loukinov ◽  
B.T. Hyman ◽  
...  
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Lipoprotein Receptor ◽  
Wild Type ◽  
Vldl Receptor ◽  
Mutant Receptor ◽  
Class A ◽  
Density Lipoprotein Receptor ◽  
Type Receptor

The very low density lipoprotein (VLDL) receptor is closely related in structure to the low density lipoprotein receptor. The ectodomain of these endocytic receptors is composed of modules which include clusters of cysteine-rich class A repeats, epidermal growth factor (EGF)-like repeats, tyrosine-tryptophan-threonine-aspartic acid (YWTD) repeats and an O-linked sugar domain. To identify important functional regions within the ectodomain of the VLDL receptor, we produced a mutant receptor in which the EGF, YWTD and O-linked sugar domains were deleted. Cells transfected with the mutant receptor were able to bind and internalize (125)I-labeled receptor associated protein (RAP). In contrast to the wild-type receptor, however, RAP did not dissociate from the mutant receptor and consequently was not degraded. Immunofluoresence data indicated that once bound to the mutant receptor, fluorescent-labeled RAP co-localized with markers of the endosomal pathway, whereas, in cells expressing the wild-type receptor, RAP fluorescence co-localized with lysosomal markers. Thus this deleted region is responsible for ligand uncoupling within the endosomes. To identify regions responsible for ligand recognition, soluble receptor fragments containing the eight cysteine-rich class A repeats were produced. (125)I-RAP and (125)I-labeled urokinase-type plasminogen activator:plasminogen activator inhibitor type I (uPA:PAI-1) complexes bound to the soluble fragment with K(D, app) values of 0.3 and 14 nM, respectively. Deletion analysis demonstrate that high affinity RAP binding requires the first four cysteine-rich class A repeats (L1-4) in the VLDL receptor while the second repeat (L2) appears responsible for binding uPA:PAI-1 complexes. Together, these results confirm that ligand uncoupling occurs via an allosteric-type mechanism in which pH induced changes in the EGF and/or YWTD repeats alter the ligand binding properties at the amino-terminal portion of the molecule.

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