Conklin BR

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Full name : Conklin Bruce R

First name : Bruce R

Mail : Department of Medicine, University of California San Francisco, San Francisco, California

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Country : USA

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References (10)

Title : Gene-trapped mouse embryonic stem cell-derived cardiac myocytes and human genetics implicate AKAP10 in heart rhythm regulation - Tingley_2007_Proc.Natl.Acad.Sci.U.S.A_104_8461
Author(s) : Tingley WG , Pawlikowska L , Zaroff JG , Kim T , Nguyen T , Young SG , Vranizan K , Kwok PY , Whooley MA , Conklin BR
Ref : Proc Natl Acad Sci U S A , 104 :8461 , 2007
Abstract : Sudden cardiac death due to abnormal heart rhythm kills 400,000-460,000 Americans each year. To identify genes that regulate heart rhythm, we are developing a screen that uses mouse embryonic stem cells (mESCs) with gene disruptions that can be differentiated into cardiac cells for phenotyping. Here, we show that the heterozygous disruption of the Akap10 (D-AKAP2) gene that disrupts the final 51 aa increases the contractile response of cultured cardiac cells to cholinergic signals. In both heterozygous and homozygous mutant mice derived from these mESCs, the same Akap10 disruption increases the cardiac response to cholinergic signals, suggesting a dominant interfering effect of the Akap10 mutant allele. The mutant mice have cardiac arrhythmias and die prematurely. We also found that a common variant of AKAP10 in humans (646V, 40% of alleles) was associated with increased basal heart rate and decreased heart rate variability (markers of low cholinergic/vagus nerve sensitivity). These markers predict an increased risk of sudden cardiac death. Although the molecular mechanism remains unknown, our findings in mutant mESCs, mice, and a common human AKAP10 SNP all suggest a role for AKAP10 in heart rhythm control. Our stem cell-based screen may provide a means of identifying other genes that control heart rhythm.
ESTHER : Tingley_2007_Proc.Natl.Acad.Sci.U.S.A_104_8461
PubMedSearch : Tingley_2007_Proc.Natl.Acad.Sci.U.S.A_104_8461
PubMedID: 17485678

Title : mGluR2 acts through inhibitory Galpha subunits to regulate transmission and long-term plasticity at hippocampal mossy fiber-CA3 synapses - Nicholls_2006_Proc.Natl.Acad.Sci.U.S.A_103_6380
Author(s) : Nicholls RE , Zhang XL , Bailey CP , Conklin BR , Kandel ER , Stanton PK
Ref : Proc Natl Acad Sci U S A , 103 :6380 , 2006
Abstract : Presynaptic inhibitory G protein-coupled receptors play a critical role in regulating transmission at a number of synapses in the central and peripheral nervous system. We generated transgenic mice that express a constitutively active form of an inhibitory Galpha subunit to examine the molecular mechanisms underlying the actions of one such receptor, metabotropic glutamate receptor (mGluR) 2, at mossy fiber-CA3 synapses in the hippocampus. mGluR2 participates in at least three types of mossy fiber synaptic plasticity, (i) transient suppression of synaptic transmission, (ii) long-term depression (LTD), and (iii) inhibition of long-term potentiation (LTP), and we find that inhibitory Galpha signaling is sufficient to account for the actions of mGluR2 in each. The fact that constitutively active Galphai2 occludes the transient suppression of synaptic transmission by mGluR2, while enhancing LTD, suggests further that these two forms of plasticity are expressed via different mechanisms. In addition, the LTP deficit observed in constitutively active Galphai2-expressing mice suggests that mGluR2 activation may serve as a metaplastic switch to permit the induction of LTD by inhibiting LTP.
ESTHER : Nicholls_2006_Proc.Natl.Acad.Sci.U.S.A_103_6380
PubMedSearch : Nicholls_2006_Proc.Natl.Acad.Sci.U.S.A_103_6380
PubMedID: 16606834

Title : Molecular basis of receptor\/G protein coupling selectivity studied by coexpression of wild type and mutant m2 muscarinic receptors with mutant G alpha(q) subunits - Kostenis_1997_Biochemistry_36_1487
Author(s) : Kostenis E , Conklin BR , Wess J
Ref : Biochemistry , 36 :1487 , 1997
Abstract : The molecular basis of receptor/G protein coupling selectivity was studied by using the m2 muscarinic receptor, a prototypical G(i/o)-coupled receptor as a model system. We could recently show that the m2 receptor can efficiently interact with mutant G protein alpha(q) subunits in which the last five amino acids were replaced with alpha(i2) or alpha(o) sequence [Liu, J., Conklin, B. R., Blin, N., Yun, J., & Wess, J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11642-11646]. Additional mutagenesis studies led to the identification of a four-amino-acid motif on the m2 receptor (Val385, Thr386, Ile389, and Leu390) that is predicted to functionally interact with the C-terminal portion of alpha(i/o) subunits. To further investigate the structural requirements for this interaction to occur, these four m2 receptor residues were replaced, either individually or in combination, with the corresponding residues present in the G(q/11)-coupled muscarinic receptors (m1, m3, and m5). The ability of the resulting mutant m2 receptors to interact with a mutant alpha(q) subunit (qo5) in which the last five amino acids were replaced with alpha(o) sequence was investigated in co-transfected COS-7 cells [studied biochemical response: stimulation of phosphatidyl inositol (PI) hydrolysis]. Our data suggest that the presence of three of the four targeted m2 receptor residues (Val385, Thr386, and Ile389) is essential for efficient recognition of C-terminal alpha(i/o) sequences. To study which specific amino acids within the C-terminal segment of alpha(i/o) subunits are critical for this interaction to occur, the wild type m2 receptor was co-expressed with a series of mutant alpha(q) subunits containing single or multiple alpha(q) --> alpha(i1,2) point mutations at their C-terminus. Remarkably, the wild type m2 receptor, while unable to efficiently stimulate wild type alpha(q), gained the ability to productively interact with three alpha(q) single-point mutants, providing the first example that the receptor coupling selectivity of G protein alpha subunits can be switched by single amino acid substitutions. Given the high degree of structural homology among different G protein-coupled receptors and among different classes of G protein alpha subunits, our results should be of broad general relevance.
ESTHER : Kostenis_1997_Biochemistry_36_1487
PubMedSearch : Kostenis_1997_Biochemistry_36_1487
PubMedID: 9063897

Title : Interactions of muscarinic receptors with the heterotrimeric G proteins Gq and G12: transduction of proliferative signals - Burstein_1997_J.Neurochem_68_525
Author(s) : Burstein ES , Brauner-Osborne H , Spalding TA , Conklin BR , Brann MR
Ref : Journal of Neurochemistry , 68 :525 , 1997
Abstract : The proliferative and transforming properties of m2 and m5 muscarinic acetylcholine receptors and a series of wild-type, chimeric, and mutant G proteins were measured alone or in combination in NIH 3T3 cells to determine which G proteins mediate these signals and to what extent these signals can be influenced by changing the stoichiometry of receptors and G proteins. Responses were measured using the focus-forming assay and a novel assay called R-SAT (Receptor Selection and Amplification Technology) in which proliferative responses are monitored using a reporter gene. Individually, GTPase-deficient mutants (*) of G alpha q and G alpha 12, wild-type G alpha q, and m5 were active in R-SAT. G alpha 12* and m5 also induced focus formation. m2 was inactive in both assays. The ability of m5 to induce foci was significantly reduced by coexpression of G alpha q*. Synergistic effects of receptor/ G protein combinations were not observed in focus-forming assays but were readily detected by R-SAT. Coexpression of G alpha q with m5 induced constitutive activity in R-SAT and increased the potency of agonists at m5 by 90-fold. G alpha q also evoked agonist-dependent responses from m2 but not constitutive activity. Agonist potency was increased 10-fold at m2 and decreased 15-fold at m5 when these receptors were coexpressed with G alpha qi5, a chimeric G protein containing the five C-terminal residues of G alpha i2, compared with coexpression with G alpha q. Both G alpha q and G alpha qi5 had biphasic effects on the proliferative responses to m5 and m2, respectively, inhibiting responses at high agonist concentrations. Coexpression of G alpha 12 or G alpha 12i5 had no effect on the concentration-response relationships of m5, but both elicited weak responses from m2. We conclude that although G alpha 12 is a more potent oncogene, G alpha q transduces m5-driven cellular responses. The demonstrations that proliferative responses can be elicited from a nonmitogenic receptor by altering the type and concentration of available G proteins and that constitutive responses can be induced by G proteins imply that both the magnitude and type of receptor-initiated signal can be regulated at the level of G proteins in vivo.
ESTHER : Burstein_1997_J.Neurochem_68_525
PubMedSearch : Burstein_1997_J.Neurochem_68_525
PubMedID: 9003037

Title : Molecular mechanisms involved in muscarinic acetylcholine receptor-mediated G protein activation studied by insertion mutagenesis - Liu_1996_J.Biol.Chem_271_6172
Author(s) : Liu J , Blin N , Conklin BR , Wess J
Ref : Journal of Biological Chemistry , 271 :6172 , 1996
Abstract : We have recently shown that a four-amino acid epitope (VTIL) on the m2 muscarinic receptor (corresponding to Val385, Thr386, Ile389, and Leu390) is essential for Gi/o coupling specificity and Gi/o activation (Liu, J., Conklin, B. R., Blin, N., Yun, J., and Wess, J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11642-11646). Because this sequence element is thought to be located at the junction between the third intracellular loop and the sixth transmembrane helix (TM VI), we speculated that agonist binding to the m2 receptor protein results in conformational changes that enable the VTIL motif to interact with Gi/o proteins. To test the hypothesis that such structural changes might involve a relative movement of TM VI toward the cytoplasm, we created a series of mutant m2 muscarinic receptors in which one to four extra Ala residues were inserted into TM VI immediately after Leu390. Based on the geometry of an alpha-helix, such mutations are predicted to "push" the VTIL sequence away from the lipid bilayer. Consistent with our working hypothesis, second messenger assays with transfected COS-7 cells showed that all mutant m2 receptors containing extra Ala residues C-terminal of Leu390 could activate the proper G proteins even in the absence of agonist. However, replacement of the VTIL motif in such constitutively active m2 receptors with the corresponding m3 muscarinic receptor sequence (AALS) or deletion of Ala391 from the wild type m2 receptor completely abolished G protein coupling. Interestingly, introduction of extra Ala residues C-terminal of the AALS motif in the m3 muscarinic receptor completely abolished functional activity. Mutant m2 and m3 receptors that contained extra Ala residues immediately N-terminal of the VTIL and AALS motif, respectively, displayed wild type-like coupling properties. Our data are consistent with a model in which agonist binding to the m2 muscarinic receptor leads to a relative movement of TM VI toward the cytoplasm, thus enabling the adjacent VTIL sequence to interact with the C terminus of Galpha(i/o) subunits.
ESTHER : Liu_1996_J.Biol.Chem_271_6172
PubMedSearch : Liu_1996_J.Biol.Chem_271_6172
PubMedID: 8626406

Title : Identification of a receptor\/G-protein contact site critical for signaling specificity and G-protein activation - Liu_1995_Proc.Natl.Acad.Sci.U.S.A_92_11642
Author(s) : Liu J , Conklin BR , Blin N , Yun J , Wess J
Ref : Proc Natl Acad Sci U S A , 92 :11642 , 1995
Abstract : Each G protein-coupled receptor recognizes only a distinct subset of the many structurally closely related G proteins expressed within a cell. How this selectively is achieved at a molecular level is not well understood, particularly since no specific point-to-point contact sites between a receptor and its cognate G protein(s) have been identified. In this study, we demonstrate that a 4-aa epitope on the m2 muscarinic acetylcholine receptor, a prototypical Gi/o-coupled receptor, can specifically recognize the C-terminal 5 aa of alpha subunits of the Gi/o protein family. The m2 receptor residues involved in this interaction are predicted to be located on one side of an alpha-helical receptor region present at the junction between the third intracellular loop and the sixth transmembrane domain. Coexpression studies with hybrid m2/m3 muscarinic receptors and mutant G-protein alpha q subunits showed that the receptor/G-protein contact site identified in this study is essential for coupling specificity and G-protein activation.
ESTHER : Liu_1995_Proc.Natl.Acad.Sci.U.S.A_92_11642
PubMedSearch : Liu_1995_Proc.Natl.Acad.Sci.U.S.A_92_11642
PubMedID: 8524820

Title : A recombinant calcitonin receptor independently stimulates 3',5'-cyclic adenosine monophosphate and Ca2+\/inositol phosphate signaling pathways - Chabre_1992_Mol.Endocrinol_6_551
Author(s) : Chabre O , Conklin BR , Lin HY , Lodish HF , Wilson E , Ives HE , Catanzariti L , Hemmings BA , Bourne HR
Ref : Mol Endocrinol , 6 :551 , 1992
Abstract : Calcitonin (CT), a polypeptide hormone, regulates calcium homeostasis by activating surface receptors coupled to stimulation of adenylyl cyclase in bone and kidney cells. CT has also been reported to increase cytoplasmic Ca2+ in osteoclasts and renal tubule cells. Signaling pathways activated by a recombinant porcine renal calcitonin receptor transiently expressed in HEK-293 cells were studied. In cells expressing the recombinant CT receptor, salmon CT stimulated cAMP accumulation (EC50, 0.16 nM) and synthesis of inositol phosphates (IP; EC50, 3.7 nM). Two other recombinant receptors, the m1-muscarinic acetylcholine receptor and the LH receptor, activated synthesis of either IP or cAMP, respectively, but not both. Stable expression of the CT receptor in a CT receptor-deficient cell line, M18, restored the cells' ability to increase cytoplasmic Ca2+ in response to salmon CT. These results show that a single recombinant CT receptor can independently activate effector pathways mediated by cAMP and IP/Ca2+.
ESTHER : Chabre_1992_Mol.Endocrinol_6_551
PubMedSearch : Chabre_1992_Mol.Endocrinol_6_551
PubMedID: 1316547

Title : Carbachol-induced reverse transformation of Chinese hamster ovary cells transfected with and expressing the m5 muscarinic acetylcholine receptor - Felder_1989_FEBS.Lett_245_75
Author(s) : Felder CC , Ma AL , Conklin BR
Ref : FEBS Letters , 245 :75 , 1989
Abstract : Reverse transformation was induced in Chinese hamster ovary (CHO) cells transfected with and stably expressing the m5 subtype of the muscarinic acetylcholine receptor when stimulated with the muscarinic agonist, carbachol. Atropine, a muscarinic antagonist, blocked the carbachol-stimulated reverse transformation. CHO cells not transfected with the muscarinic receptor did not change with added carbachol. PMA induced reverse transformation without increasing cAMP accumulation in CHO cells. Carbachol, prostaglandin E2, and cholecystokinin increased cAMP accumulation but only carbachol caused reverse transformation. Carbachol-stimulated cAMP accumulation occurred at a higher concentration (EC50 10 microM) than did carbachol-stimulated reverse transformation (EC50 63 nM). Muscarinic m5 acetylcholine receptor transfected into CHO cells can induce reverse transformation which may be independent of cAMP.
ESTHER : Felder_1989_FEBS.Lett_245_75
PubMedSearch : Felder_1989_FEBS.Lett_245_75
PubMedID: 2466702

Title : Poster: Carbachol stimulation causes inhibition of mitogenesis and cell elongation in CHO cells transfected with muscarinrc receptor genes -
Author(s) : Conklin BR , Brann MR , Buckley NJ , Bonner TI , Ma AL , Felder CC , Axelrod J
Ref : Trends in Pharmacological Sciences , Suppl :117 , 1989
PubMedID:

Title : Stimulation of arachidonic acid release and inhibition of mitogenesis by cloned genes for muscarinic receptor subtypes stably expressed in A9 L cells - Conklin_1988_Proc.Natl.Acad.Sci.U.S.A_85_8698
Author(s) : Conklin BR , Brann MR , Buckley NJ , Ma AL , Bonner TI , Axelrod J
Ref : Proc Natl Acad Sci U S A , 85 :8698 , 1988
Abstract : A family of genes encoding four distinct muscarinic receptors (designated m1-m4) has been cloned and stably expressed in A9 L cells. When the m1 and m3 receptors were stimulated with carbachol, there was a rapid rise of liberated arachidonic acid, inositol phosphates, and cAMP, while m2 and m4 receptor stimulation had no detectable stimulation of these second messengers. Pretreatment with phorbol 12-myristate 13-acetate (PMA) caused a marked acceleration and amplification of m1 and m3 receptor-mediated arachidonic acid release. In contrast, m1- and m3-mediated inositol phosphate formation was inhibited by the same PMA pretreatment. Arachidonic acid release was unaffected by manipulations of cAMP levels. Arachidonic acid production was inhibited by calcium-free medium and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8; an inhibitor of cytosolic calcium mobilization) yet was unaffected by verapamil, a calcium-channel blocker. These experiments show that arachidonic acid release induced by the m1 and m3 receptors is regulated independently of phospholipase C and cAMP accumulation. Carbachol stimulation of the m1 and m3 cAMP accumulation. Carbachol stimulation of the m1 and m3 receptors also markedly decreased mitogenesis as measured by thymidine incorporation. The m1 receptor-mediated inhibition of mitogenesis could be partially blocked by indomethacin, a cyclooxygenase inhibitor. The inhibition of mitogenesis could be mimicked by cAMP elevation.
ESTHER : Conklin_1988_Proc.Natl.Acad.Sci.U.S.A_85_8698
PubMedSearch : Conklin_1988_Proc.Natl.Acad.Sci.U.S.A_85_8698
PubMedID: 2847172