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1.
Proc Natl Acad Sci U S A ; 121(16): e2316651121, 2024 Apr 16.
Article in English | MEDLINE | ID: mdl-38588418

ABSTRACT

Protecting chromosome ends from misrecognition as double-stranded (ds) DNA breaks is fundamental to eukaryotic viability. The protein complex shelterin prevents a DNA damage response at mammalian telomeres. Mammalian shelterin proteins TRF1 and TRF2 and their homologs in yeast and protozoa protect telomeric dsDNA. N-terminal homodimerization and C-terminal Myb-domain-mediated dsDNA binding are two structural hallmarks of end protection by TRF homologs. Yet our understanding of how Caenorhabditis elegans protects its telomeric dsDNA is limited. Recently identified C. elegans proteins TEBP-1 (also called DTN-1) and TEBP-2 (also called DTN-2) are functional homologs of TRF proteins, but how they bind DNA and whether or how they dimerize is not known. TEBP-1 and TEBP-2 harbor three Myb-containing domains (MCDs) and no obvious dimerization domain. We demonstrate biochemically that only the third MCD binds DNA. We solve the X-ray crystal structure of TEBP-2 MCD3 with telomeric dsDNA to reveal the structural mechanism of telomeric dsDNA protection in C. elegans. Mutagenesis of the DNA-binding site of TEBP-1 and TEBP-2 compromises DNA binding in vitro, and increases DNA damage signaling, lengthens telomeres, and decreases brood size in vivo. Via an X-ray crystal structure, biochemical validation of the dimerization interface, and SEC-MALS analysis, we demonstrate that MCD1 and MCD2 form a composite dimerization module that facilitates not only TEBP-1 and TEBP-2 homodimerization but also heterodimerization. These findings provide fundamental insights into C. elegans telomeric dsDNA protection and highlight how different eukaryotes have evolved distinct strategies to solve the chromosome end protection problem.


Subject(s)
Caenorhabditis elegans Proteins , Telomere-Binding Proteins , Animals , Telomere-Binding Proteins/metabolism , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Dimerization , Telomeric Repeat Binding Protein 1/genetics , Telomeric Repeat Binding Protein 1/chemistry , Telomeric Repeat Binding Protein 1/metabolism , Protein Binding , Telomere/genetics , Telomere/metabolism , Shelterin Complex , DNA/metabolism , Telomeric Repeat Binding Protein 2 , Mammals/genetics
2.
Science ; 381(6659): 771-778, 2023 08 18.
Article in English | MEDLINE | ID: mdl-37590346

ABSTRACT

Protection of telomeres 1 (POT1) is the 3' single-stranded overhang-binding telomeric protein that prevents an ataxia telangiectasia and Rad3-related (ATR) DNA damage response (DDR) at chromosome ends. What precludes the DDR machinery from accessing the telomeric double-stranded-single-stranded junction is unknown. We demonstrate that human POT1 binds this junction by recognizing the phosphorylated 5' end of the chromosome. High-resolution crystallographic structures reveal that the junction is capped by POT1 through a "POT-hole" surface, the mutation of which compromises junction protection in vitro and telomeric 5'-end definition and DDR suppression in human cells. Whereas both mouse POT1 paralogs bind the single-stranded overhang, POT1a, not POT1b, contains a POT-hole and binds the junction, which explains POT1a's sufficiency for end protection. Our study shifts the paradigm for DDR suppression at telomeres by highlighting the importance of protecting the double-stranded-single-stranded junction.


Subject(s)
DNA , Shelterin Complex , Telomere-Binding Proteins , Telomere , Animals , Humans , Mice , Crystallography , DNA/chemistry , DNA/metabolism , Mutation , Shelterin Complex/chemistry , Shelterin Complex/genetics , Shelterin Complex/metabolism , Telomere/chemistry , Telomere/metabolism , Telomere-Binding Proteins/chemistry , Telomere-Binding Proteins/genetics , Telomere-Binding Proteins/metabolism
3.
Life Sci Alliance ; 6(6)2023 06.
Article in English | MEDLINE | ID: mdl-36963832

ABSTRACT

Telomerase processively adds telomeric DNA repeats to chromosome ends using catalytic protein subunit TERT and a template on its RNA subunit TR. Mammalian telomerase is recruited to telomeres by the TEL patch and NOB regions of shelterin component TPP1. Recent cryo-EM structures of human telomerase reveal that a composite TERT TEN-(IFD-TRAP) domain interacts with TPP1. Here, we generate TERT mutants to demonstrate that a three-way TEN-(IFD-TRAP)-TPP1 interaction is critical for telomerase recruitment to telomeres and processive telomere repeat addition. Single mutations of IFD-TRAP at its interface with TR or the DNA primer impair telomerase catalysis. We further reveal the importance of TERT motif 3N and TEN domain loop 99FGF101 in telomerase action. Finally, we demonstrate that TPP1 TEL patch loop residue F172, which undergoes a structural rearrangement to bind telomerase, contributes to the human-mouse species specificity of the telomerase-TPP1 interaction. Our study provides insights into the multiple functions of TERT IFD-TRAP, reveals novel TERT and TPP1 elements critical for function, and helps explain how TPP1 binding licenses robust telomerase action at natural chromosome ends.


Subject(s)
Telomerase , Animals , Humans , Mice , Telomerase/metabolism , Telomere-Binding Proteins/genetics , Telomere-Binding Proteins/metabolism , Shelterin Complex , Telomere/genetics , Telomere/metabolism , Mutation , Mammals/genetics
4.
Nat Commun ; 12(1): 5514, 2021 09 17.
Article in English | MEDLINE | ID: mdl-34535663

ABSTRACT

Human shelterin components POT1 and TPP1 form a stable heterodimer that protects telomere ends from ATR-dependent DNA damage responses and regulates telomerase-dependent telomere extension. Mice possess two functionally distinct POT1 proteins. POT1a represses ATR/CHK1 DNA damage responses and the alternative non-homologous end-joining DNA repair pathway while POT1b regulates C-strand resection and recruits the CTC1-STN1-TEN1 (CST) complex to telomeres to mediate C-strand fill-in synthesis. Whether POT1a and POT1b are involved in regulating the length of the telomeric G-strand is unclear. Here we demonstrate that POT1b, independent of its CST function, enhances recruitment of telomerase to telomeres through three amino acids in its TPP1 interacting C-terminus. POT1b thus coordinates the synthesis of both telomeric G- and C-strands. In contrast, POT1a negatively regulates telomere length by inhibiting telomerase recruitment to telomeres. The identification of unique amino acids between POT1a and POT1b helps us understand mechanistically how human POT1 switches between end protective functions and promoting telomerase recruitment.


Subject(s)
DNA-Binding Proteins/metabolism , Telomerase/metabolism , Telomere-Binding Proteins/metabolism , Telomere/metabolism , Animals , CRISPR-Cas Systems/genetics , DNA Mutational Analysis , Mice , Protein Binding , Rad51 Recombinase/metabolism , Sarcoma/pathology
5.
JCI Insight ; 6(9)2021 05 10.
Article in English | MEDLINE | ID: mdl-33822766

ABSTRACT

Telomerase catalyzes chromosome end replication in stem cells and other long-lived cells. Mutations in telomerase or telomere-related genes result in diseases known as telomeropathies. Telomerase is recruited to chromosome ends by the ACD/TPP1 protein (TPP1 hereafter), a component of the shelterin complex that protects chromosome ends from unwanted end joining. TPP1 facilitates end protection by binding shelterin proteins POT1 and TIN2. TPP1 variants have been associated with telomeropathies but remain poorly characterized in vivo. Disease variants and mutagenesis scans provide efficient avenues to interrogate the distinct physiological roles of TPP1. Here, we conduct mutagenesis in the TIN2- and POT1-binding domains of TPP1 to discover mutations that dissect TPP1's functions. Our results extend current structural data to reveal that the TPP1-TIN2 interface is more extensive than previously thought and highlight the robustness of the POT1-TPP1 interface. Introduction of separation-of-function mutants alongside known TPP1 telomeropathy mutations in mouse hematopoietic stem cells (mHSCs) lacking endogenous TPP1 demonstrated a clear phenotypic demarcation. TIN2- and POT1-binding mutants were unable to rescue mHSC failure resulting from end deprotection. In contrast, TPP1 telomeropathy mutations sustained mHSC viability, consistent with their selectively impacting end replication. These results highlight the power of scanning mutagenesis in revealing structural interfaces and dissecting multifunctional genes.


Subject(s)
Hematopoiesis/genetics , Hematopoietic Stem Cells/metabolism , Shelterin Complex/metabolism , Telomere-Binding Proteins/genetics , Animals , Cell Survival/genetics , Humans , Mice , Mutagenesis, Site-Directed , Shelterin Complex/genetics , Telomere-Binding Proteins/metabolism , Telomeric Repeat Binding Protein 2/metabolism
6.
Proc Natl Acad Sci U S A ; 116(52): 26505-26515, 2019 Dec 26.
Article in English | MEDLINE | ID: mdl-31822618

ABSTRACT

Telomerase catalyzes telomeric DNA synthesis at chromosome ends to allow for continued cell division. The telomeric protein TPP1 is essential for enhancing the processivity of telomerase and recruiting the enzyme to telomeres. The telomerase interaction surface on human TPP1 has been mapped to 2 regions of the N-terminal oligosaccharide/oligonucleotide-binding (OB) domain, namely the TPP1 glutamate (E) and leucine (L)-rich (TEL) patch and the N terminus of TPP1-oligosaccharide/oligonucleotide-binding (NOB) region. To map the telomerase side of the interface, we exploited the predicted structural similarities for human and Tetrahymena thermophila telomerase as well as the species specificity of human and mouse telomerase for their cognate TPP1 partners. We show that swapping in the telomerase essential N-terminal (TEN) and insertions in fingers domain (IFD)-TRAP regions of the human telomerase catalytic protein subunit TERT into the mouse TERT backbone is sufficient to bias the species specificity toward human TPP1. Employing a structural homology-based mutagenesis screen focused on surface residues of the TEN and IFD regions, we identified TERT residues that are critical for contacting TPP1 but dispensable for other aspects of telomerase structure or function. We present a functionally validated structural model for how human telomerase engages TPP1 at telomeres, setting the stage for a high-resolution structure of this interface.

7.
Cell Rep ; 27(12): 3511-3521.e7, 2019 06 18.
Article in English | MEDLINE | ID: mdl-31216472

ABSTRACT

Telomerase replicates chromosome ends in germ and somatic stem cells to facilitate their continued proliferation. Telomerase action depends on the telomeric protein TPP1, which recruits telomerase to telomeres and facilitates processive DNA synthesis. Here, we identify separation-of-function long (TPP1-L) and short (TPP1-S) isoforms of TPP1 that appear to be generated from separate transcripts and differ only in 86 amino acids at their N terminus. Although both isoforms retain the ability to recruit telomerase, only TPP1-S facilitates efficient telomere synthesis. We find that TPP1-S is the predominant isoform in somatic cells, and strikingly, TPP1-L is the major isoform in differentiated male germ cells. We observed that TERT expression persists in these germ cells, suggesting that TPP1-L could restrain telomerase in this context. We show how differential expression of TPP1 isoforms determines telomerase function and demonstrate how alternative transcription start sites allow one gene to perform distinct functions in different biological contexts.


Subject(s)
Aminopeptidases/metabolism , Chromosomes, Human/metabolism , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , Germ Cells/metabolism , Serine Proteases/metabolism , Shelterin Complex , Telomerase/metabolism , Telomere Homeostasis , Telomere-Binding Proteins , Testis/metabolism , Amino Acid Sequence , Aminopeptidases/genetics , Chromosomes, Human/genetics , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics , Germ Cells/cytology , HeLa Cells , Humans , Male , Protein Binding , Protein Isoforms , Sequence Homology , Serine Proteases/genetics , Shelterin Complex/genetics , Shelterin Complex/metabolism , Telomerase/genetics , Telomere-Binding Proteins/genetics , Telomere-Binding Proteins/metabolism , Testis/cytology
8.
Cell Rep ; 22(5): 1132-1140, 2018 01 30.
Article in English | MEDLINE | ID: mdl-29386102

ABSTRACT

Telomerase recruitment to telomeres and enzymatic processivity are mediated by TPP1, an essential component of telomere integrity and telomerase function. A surface on the OB domain of TPP1 called the TEL patch is critical for TPP1's telomerase-associated functions. Here, we identify a separate region in the N terminus of the OB domain (termed NOB) of TPP1 that, like the TEL patch, is essential for telomerase repeat addition processivity in vitro as well as telomerase recruitment to telomeres and telomere lengthening in cells. Although well-conserved among most mammalian TPP1 homologs, the NOB region in mice is distinct. Swapping the sequence of human NOB into mouse TPP1 allows it to stimulate human telomerase, qualifying NOB as an important determinant of species specificity for TPP1-telomerase interaction. Our studies show that TPP1 NOB is critical for telomerase function and demonstrate that the telomerase interaction surface on TPP1 is more elaborate than previously appreciated.


Subject(s)
Telomerase/metabolism , Telomere Homeostasis/physiology , Telomere-Binding Proteins/chemistry , Telomere-Binding Proteins/metabolism , Telomere/metabolism , Animals , Chimera , Humans , Mice , Models, Molecular , Protein Domains , Shelterin Complex
9.
Nat Struct Mol Biol ; 24(12): 1064-1072, 2017 Dec.
Article in English | MEDLINE | ID: mdl-29083414

ABSTRACT

Tethering telomeres to the inner nuclear membrane (INM) allows homologous chromosome pairing during meiosis. The meiosis-specific protein TERB1 binds the telomeric protein TRF1 to establish telomere-INM connectivity and is essential for mouse fertility. Here we solve the structure of the human TRF1-TERB1 interface to reveal the structural basis for telomere-INM linkage. Disruption of this interface abrogates binding and compromises telomere-INM attachment in mice. An embedded CDK-phosphorylation site within the TRF1-binding region of TERB1 provides a mechanism for cap exchange, a late-pachytene phenomenon involving the dissociation of the TRF1-TERB1 complex. Indeed, further strengthening this interaction interferes with cap exchange. Finally, our biochemical analysis implicates distinct complexes for telomere-INM tethering and chromosome-end protection during meiosis. Our studies unravel the structure, stoichiometry, and physiological implications underlying telomere-INM tethering, thereby providing unprecedented insights into the unique function of telomeres in meiosis.


Subject(s)
Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Meiosis/physiology , Nuclear Envelope/metabolism , Telomere/metabolism , Telomeric Repeat Binding Protein 1/metabolism , Animals , Binding Sites/genetics , Carrier Proteins/genetics , Cell Cycle Proteins/genetics , Chromosome Pairing , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , Protein Binding/physiology , rap1 GTP-Binding Proteins/metabolism
10.
Proc Natl Acad Sci U S A ; 113(46): 13021-13026, 2016 11 15.
Article in English | MEDLINE | ID: mdl-27807141

ABSTRACT

Telomerase replicates chromosome ends to facilitate continued cell division. Mutations that compromise telomerase function result in stem cell failure diseases, such as dyskeratosis congenita (DC). One such mutation (K170Δ), residing in the telomerase-recruitment factor TPP1, provides an excellent opportunity to structurally, biochemically, and genetically dissect the mechanism of such diseases. We show through site-directed mutagenesis and X-ray crystallography that this TPP1 disease mutation deforms the conformation of two critical amino acids of the TEL [TPP1's glutamate (E) and leucine-rich (L)] patch, the surface of TPP1 that binds telomerase. Using CRISPR-Cas9 technology, we demonstrate that introduction of this mutation in a heterozygous manner is sufficient to shorten telomeres in human cells. Our findings rule out dominant-negative effects of the mutation. Instead, these findings implicate reduced TEL patch dosage in causing telomere shortening. Our studies provide mechanistic insight into telomerase-deficiency diseases and encourage the development of gene therapies to counter such diseases.


Subject(s)
Dyskeratosis Congenita/genetics , Telomere-Binding Proteins/chemistry , Telomere-Binding Proteins/genetics , Cell Line, Tumor , Crystallography, X-Ray , HEK293 Cells , Humans , Mutagenesis, Site-Directed , Mutation , Protein Conformation , Shelterin Complex , Telomerase/metabolism , Telomere/metabolism
11.
Structure ; 21(3): 438-48, 2013 Mar 05.
Article in English | MEDLINE | ID: mdl-23434405

ABSTRACT

The heterotrimeric G protein Gαq is a key regulator of blood pressure, and excess Gαq signaling leads to hypertension. A specific inhibitor of Gαq is the GTPase activating protein (GAP) known as regulator of G protein signaling 2 (RGS2). The molecular basis for how Gαq/11 subunits serve as substrates for RGS proteins and how RGS2 mandates its selectivity for Gαq is poorly understood. In crystal structures of the RGS2-Gαq complex, RGS2 docks to Gαq in a different orientation from that observed in RGS-Gαi/o complexes. Despite its unique pose, RGS2 maintains canonical interactions with the switch regions of Gαq in part because its α6 helix adopts a distinct conformation. We show that RGS2 forms extensive interactions with the α-helical domain of Gαq that contribute to binding affinity and GAP potency. RGS subfamilies that do not serve as GAPs for Gαq are unlikely to form analogous stabilizing interactions.


Subject(s)
GTP-Binding Protein alpha Subunits, Gq-G11/chemistry , Molecular Dynamics Simulation , Protein Subunits/chemistry , RGS Proteins/chemistry , Amino Acid Sequence , Animals , Binding Sites , Cattle , Crystallography, X-Ray , Escherichia coli/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , Humans , Mice , Molecular Docking Simulation , Molecular Sequence Data , Mutation , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Protein Subunits/genetics , RGS Proteins/genetics , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Sequence Alignment , Structure-Activity Relationship
12.
Structure ; 20(8): 1300-9, 2012 Aug 08.
Article in English | MEDLINE | ID: mdl-22727813

ABSTRACT

Cardiovascular homeostasis is maintained in part by the rapid desensitization of activated heptahelical receptors that have been phosphorylated by G protein-coupled receptor kinase 2 (GRK2). However, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. We have determined crystallographic structures of GRK2 bound to an RNA aptamer that potently and selectively inhibits kinase activity. Key to the mechanism of inhibition is the positioning of an adenine nucleotide into the ATP-binding pocket and interactions with the basic αF-αG loop region of the GRK2 kinase domain. Constraints imposed on the RNA by the terminal stem of the aptamer also play a role. These results highlight how a high-affinity aptamer can be used to selectively trap a novel conformational state of a protein kinase.


Subject(s)
Aptamers, Nucleotide/chemistry , G-Protein-Coupled Receptor Kinase 2/chemistry , Amino Acid Motifs , Animals , Base Sequence , Catalytic Domain , Cattle , Crystallography, X-Ray , G-Protein-Coupled Receptor Kinase 2/antagonists & inhibitors , Hydrogen Bonding , Models, Molecular , Nucleic Acid Conformation , Protein Binding , Protein Structure, Quaternary
13.
Proc Natl Acad Sci U S A ; 108(37): E667-73, 2011 Sep 13.
Article in English | MEDLINE | ID: mdl-21876134

ABSTRACT

Few experimental techniques can assess the orientation of peripheral membrane proteins in their native environment. Sum Frequency Generation (SFG) vibrational spectroscopy was applied to study the formation of the complex between G protein-coupled receptor (GPCR) kinase 2 (GRK2) and heterotrimeric G protein ß(1)γ(2) subunits (Gßγ) at a lipid bilayer, without any exogenous labels. The most likely membrane orientation of the GRK2-Gßγ complex differs from that predicted from the known protein crystal structure, and positions the predicted receptor docking site of GRK2 such that it would more optimally interact with GPCRs. Gßγ also appears to change its orientation after binding to GRK2. The developed methodology is widely applicable for the study of other membrane proteins in situ.


Subject(s)
G-Protein-Coupled Receptor Kinase 2/chemistry , G-Protein-Coupled Receptor Kinase 2/metabolism , GTP-Binding Protein beta Subunits/chemistry , GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/chemistry , GTP-Binding Protein gamma Subunits/metabolism , Membranes, Artificial , Lipid Bilayers/chemistry , Models, Molecular , Phosphatidylcholines/chemistry , Phosphatidylglycerols/chemistry , Protein Binding , Protein Conformation , Spectrum Analysis , Vibration
14.
Nat Struct Mol Biol ; 18(9): 999-1005, 2011 Aug 07.
Article in English | MEDLINE | ID: mdl-21822282

ABSTRACT

The enzyme phospholipase C-ß (PLCß) is a crucial regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to members of the Gq family of heterotrimeric G proteins. We have determined atomic structures of two invertebrate homologs of PLCß (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLCß3 considerably increase basal activity and diminish stimulation by Gαq. Gαq binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLCß.


Subject(s)
GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Loligo/enzymology , Phospholipase C beta/chemistry , Sepia/enzymology , Animals , Crystallography, X-Ray , Models, Molecular , Mutagenesis, Site-Directed , Phospholipase C beta/physiology , Protein Structure, Secondary/physiology , Protein Structure, Tertiary
15.
J Med Chem ; 53(4): 1867-70, 2010 Feb 25.
Article in English | MEDLINE | ID: mdl-20128603

ABSTRACT

G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined crystal structures of human GRK2 in complex with Gbetagamma in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.


Subject(s)
Azepines/chemistry , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , G-Protein-Coupled Receptor Kinase 2/chemistry , GTP-Binding Protein beta Subunits/chemistry , GTP-Binding Protein gamma Subunits/chemistry , Hydroxybenzoates/chemistry , Models, Molecular , Crystallography, X-Ray , G-Protein-Coupled Receptor Kinase 2/antagonists & inhibitors , Humans , Phosphorylation , Protein Binding , Protein Conformation , Tubulin/chemistry
16.
J Biol Chem ; 283(50): 34923-34, 2008 Dec 12.
Article in English | MEDLINE | ID: mdl-18936096

ABSTRACT

Transmembrane signaling through G alpha(q)-coupled receptors is linked to physiological processes such as cardiovascular development and smooth muscle function. Recent crystallographic studies have shown how G alpha(q) interacts with two activation-dependent targets, p63RhoGEF and G protein-coupled receptor kinase 2 (GRK2). These proteins bind to the effector-binding site of G alpha(q) in a manner that does not appear to physically overlap with the site on G alpha(q) bound by regulator of G-protein signaling (RGS) proteins, which function as GTPase-activating proteins (GAPs). Herein we confirm the formation of RGS-G alpha(q)-GRK2/p63RhoGEF ternary complexes using flow cytometry protein interaction and GAP assays. RGS2 and, to a lesser extent, RGS4 are negative allosteric modulators of Galpha(q) binding to either p63RhoGEF or GRK2. Conversely, GRK2 enhances the GAP activity of RGS4 but has little effect on that of RGS2. Similar but smaller magnitude responses are induced by p63RhoGEF. The fact that GRK2 and p63RhoGEF respond similarly to these RGS proteins supports the hypothesis that GRK2 is a bona fide G alpha(q) effector. The results also suggest that signal transduction pathways initiated by GRK2, such as the phosphorylation of G protein-coupled receptors, and by p63RhoGEF, such as the activation of gene transcription, can be regulated by RGS proteins via both allosteric and GAP mechanisms.


Subject(s)
G-Protein-Coupled Receptor Kinase 2/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Guanine Nucleotide Exchange Factors/metabolism , RGS Proteins/chemistry , Allosteric Site , Binding, Competitive , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Flow Cytometry/methods , GTP Phosphohydrolases/metabolism , Humans , Kinetics , Molecular Conformation , Protein Binding , Protein Structure, Tertiary , Rho Guanine Nucleotide Exchange Factors
17.
J Biol Chem ; 281(24): 16785-93, 2006 Jun 16.
Article in English | MEDLINE | ID: mdl-16613860

ABSTRACT

We describe the 2.6-A crystal structure of human G protein-coupled receptor kinase (GRK)-6, a key regulator of dopaminergic signaling and lymphocyte chemotaxis. GRK6 is a member of the GRK4 subfamily of GRKs, which is represented in most, if not all, metazoans. Comparison of GRK6 with GRK2 confirms that the catalytic core of all GRKs consists of intimately associated kinase and regulator of G protein signaling (RGS) homology domains. Despite being in complex with an ATP analog, the kinase domain of GRK6 remains in an open, presumably inactive conformation, suggesting that G protein-coupled receptors activate GRKs by inducing kinase domain closure. The structure reveals a putative phospholipid-binding site near the N terminus of GRK6 and structural elements within the kinase substrate channel that likely influence G protein-coupled receptor access and specificity. The crystalline GRK6 RGS homology domain forms an extensive dimer interface using conserved hydrophobic residues distinct from those in GRK2 that bind Galpha(q), although dimerization does not appear to occur in solution and is not required for receptor phosphorylation.


Subject(s)
Protein Serine-Threonine Kinases/physiology , Adenosine Triphosphate/chemistry , Animals , Binding Sites , Cell Lineage , Crystallography, X-Ray , Dimerization , G-Protein-Coupled Receptor Kinases , Humans , Insecta , Molecular Conformation , Phosphorylation , Protein Conformation , Protein Serine-Threonine Kinases/metabolism
18.
Science ; 310(5754): 1686-90, 2005 Dec 09.
Article in English | MEDLINE | ID: mdl-16339447

ABSTRACT

G protein-coupled receptor kinase 2 (GRK2) plays a key role in the desensitization of G protein-coupled receptor signaling by phosphorylating activated heptahelical receptors and by sequestering heterotrimeric G proteins. We report the atomic structure of GRK2 in complex with Galphaq and Gbetagamma, in which the activated Galpha subunit of Gq is fully dissociated from Gbetagamma and dramatically reoriented from its position in the inactive Galphabetagamma heterotrimer. Galphaq forms an effector-like interaction with the GRK2 regulator of G protein signaling (RGS) homology domain that is distinct from and does not overlap with that used to bind RGS proteins such as RGS4.


Subject(s)
GTP-Binding Protein alpha Subunits, Gq-G11/chemistry , GTP-Binding Protein beta Subunits/chemistry , GTP-Binding Protein gamma Subunits/chemistry , beta-Adrenergic Receptor Kinases/chemistry , Amino Acid Sequence , Amino Acid Substitution , Animals , Cattle , Crystallography, X-Ray , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/metabolism , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Mutation , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits/chemistry , RGS Proteins/metabolism , Signal Transduction , beta-Adrenergic Receptor Kinases/metabolism
19.
Virus Res ; 89(1): 53-63, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12367750

ABSTRACT

To examine the mechanism of HIV-1 regulation by NF-IL6 in activated human cells, we selected a Jurkat cell line that did not contain endogenous NF-IL6. In this cellular environment, we evaluated the effect of exogenous NF-IL6 on transcription mediated by native and deleted LTR sequences. In Jurkat cells stimulated with LPS and PMA, LTR-mediated transcription was enhanced by NF-IL6. The results of deletion studies revealed a central role for the basal LTR region and the TATA element in the LTR, in upregulation of reporter gene expression by NF-IL6 in activated cells. In the selected cellular environment, regulation of transcription by NF-IL6 was not evident in studies of promoter regions of other genes. The results implied that the basal region of HIV-1 LTR includes molecular properties that support activation of HIV-1 by NF-IL6 in stimulated cells.


Subject(s)
CCAAT-Enhancer-Binding Protein-beta/pharmacology , Gene Expression Regulation, Viral , HIV-1/metabolism , Jurkat Cells/virology , Transcription, Genetic/drug effects , HIV Long Terminal Repeat/genetics , HIV-1/genetics , Humans , Lymphocyte Activation , Promoter Regions, Genetic
20.
Virology ; 299(2): 256-265, 2002 Aug 01.
Article in English | MEDLINE | ID: mdl-12202228

ABSTRACT

DNA-binding and functional assays examined the role played by NF-IL6 in regulation of HIV-1 transcription in human monocyte/macrophages (U937 cells), stimulated with LPS+PMA. When incubated with nuclear extracts from stimulated cells, a region (-189/-147), containing the major NF-IL6-binding sequence and the USF site, interacted selectively with USF1 and USF2. Anti-C/EBPbeta reacted poorly with the complexes produced with the wild-type probe. In contrast, complex formation with NF-IL6 was clearly evident in experiments analyzing a probe containing an insertion in the USF site. In functional assays, increasing concentrations of a decoy against NF-IL6 reduced gene expression from the LTR of the wild-type HIV-1 variant, supporting a critical role for NF-IL6 in regulation of HIV-1 transcription in stimulated monocyte/macrophages. The decoy also reduced gene expression from a deletion construct lacking NF-IL6-binding sequences. The results implied that in LPS+PMA-stimulated monocyte/macrophages, the endogenous NF-IL6 could act via a site-independent pathway in upregulation of HIV-1 transcription. Analysis of a short DNA segment, containing the -189/-147 region, suggested functional interactions of NF-IL6 and USF. In activated cells exogenous NF-IL6 enhanced dramatically gene expression through a short DNA segment containing the NF-kappaB sites, supporting functional interactions of NF-IL6 and NF-kappaB.


Subject(s)
HIV-1/genetics , Macrophages/virology , Monocytes/virology , Transcription, Genetic , CCAAT-Enhancer-Binding Protein-beta/pharmacology , HIV Long Terminal Repeat , Humans , Lipopolysaccharides/pharmacology , Macrophage Activation , Tetradecanoylphorbol Acetate/pharmacology , U937 Cells
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