A novel mutation in the switch 3 region of G(s)α in a patient with albright hereditary osteodystrophy impairs GDP binding and receptor activation

Dennis R. Warner, Gezhi Weng, Shuhua Yu, Reuben Matalon, Lee S. Weinstein

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Abstract

Albright hereditary osteodystrophy (AHO), a disorder characterized by skeletal abnormalities and obesity, is associated with heterozygous inactivating mutations in the gene for G(s)α. A novel G(s)α mutation encoding the substitution of tryptophan for a nonconserved arginine within the switch 3 region (G(s)α R258W) was identified in an AHO patient. Although reverse transcription-polymerase chain reaction studies demonstrated that mRNA expression from wild type and mutant alleles was similar, G(s)α expression in erythrocyte membranes from the affected patient was reduced by 50%. A G(s)α R258W cDNA, as well as one with arginine replaced by alanine (G(s)α R258A), was generated, and the biochemical properties of in vitro transcription/translation products were examined. When reconstituted with cyc- membranes, both mutant proteins were able to stimulate adenylyl cyclase normally in the presence of guanosine-5'-O-(3-thiotriphosphate) (GTPγS) but had decreased ability in the presence of isoproterenol or AlF4 (a mixture of 10 μM AlCl3 and 10 mM NaF). The ability of each mutant to bind and be activated by GTPγS or AlF4/- was assessed by trypsin protection assays. Both mutants were protected normally by GTPγS but showed reduced protection in the presence of AlF4/-. The addition of excess GDP (2 mM) was able to rescue the ability of AlF4/- to protect the mutants, suggesting that they might have reduced affinity for GDP. A G(s)α R258A mutant purified from Escherichia coli had decreased affinity for GDP and an apparent rate of GDP release that was 10-fold greater than that of wild type G(s)α. Sucrose density gradient analysis demonstrated that both G(s)α R258W and G(s)α R258A were thermolabile at higher temperatures and that denaturation of both mutants was prevented by the presence of 0.1 mM GTPγS or 2 mM GDP. The crystal structure of G(s)α demonstrates that Arg258 interacts with a conserved residue in the helical domain (Gln170). Arg258 substitutions would be predicted to open the cleft between the GTPase and helical domains, allowing for increased GDP release in the inactive state, resulting in enhanced thermolability and reduced AlF4/--induced adenylyl cyclase stimulation and trypsin protection, since activation by AlF4/- requires bound GDP.

Original languageEnglish (US)
Pages (from-to)23976-23983
Number of pages8
JournalJournal of Biological Chemistry
Volume273
Issue number37
DOIs
StatePublished - Sep 11 1998

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Chemical activation
Switches
Mutation
Transcription
Adenylyl Cyclases
Trypsin
Arginine
Substitution reactions
Guanosine 5'-O-(3-Thiotriphosphate)
Membranes
Denaturation
GTP Phosphohydrolases
Polymerase chain reaction
Erythrocyte Membrane
Mutant Proteins
Isoproterenol
Tryptophan
Alanine
Escherichia coli
Reverse Transcription

ASJC Scopus subject areas

  • Biochemistry

Cite this

A novel mutation in the switch 3 region of G(s)α in a patient with albright hereditary osteodystrophy impairs GDP binding and receptor activation. / Warner, Dennis R.; Weng, Gezhi; Yu, Shuhua; Matalon, Reuben; Weinstein, Lee S.

In: Journal of Biological Chemistry, Vol. 273, No. 37, 11.09.1998, p. 23976-23983.

Research output: Contribution to journalArticle

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abstract = "Albright hereditary osteodystrophy (AHO), a disorder characterized by skeletal abnormalities and obesity, is associated with heterozygous inactivating mutations in the gene for G(s)α. A novel G(s)α mutation encoding the substitution of tryptophan for a nonconserved arginine within the switch 3 region (G(s)α R258W) was identified in an AHO patient. Although reverse transcription-polymerase chain reaction studies demonstrated that mRNA expression from wild type and mutant alleles was similar, G(s)α expression in erythrocyte membranes from the affected patient was reduced by 50{\%}. A G(s)α R258W cDNA, as well as one with arginine replaced by alanine (G(s)α R258A), was generated, and the biochemical properties of in vitro transcription/translation products were examined. When reconstituted with cyc- membranes, both mutant proteins were able to stimulate adenylyl cyclase normally in the presence of guanosine-5'-O-(3-thiotriphosphate) (GTPγS) but had decreased ability in the presence of isoproterenol or AlF4 (a mixture of 10 μM AlCl3 and 10 mM NaF). The ability of each mutant to bind and be activated by GTPγS or AlF4/- was assessed by trypsin protection assays. Both mutants were protected normally by GTPγS but showed reduced protection in the presence of AlF4/-. The addition of excess GDP (2 mM) was able to rescue the ability of AlF4/- to protect the mutants, suggesting that they might have reduced affinity for GDP. A G(s)α R258A mutant purified from Escherichia coli had decreased affinity for GDP and an apparent rate of GDP release that was 10-fold greater than that of wild type G(s)α. Sucrose density gradient analysis demonstrated that both G(s)α R258W and G(s)α R258A were thermolabile at higher temperatures and that denaturation of both mutants was prevented by the presence of 0.1 mM GTPγS or 2 mM GDP. The crystal structure of G(s)α demonstrates that Arg258 interacts with a conserved residue in the helical domain (Gln170). Arg258 substitutions would be predicted to open the cleft between the GTPase and helical domains, allowing for increased GDP release in the inactive state, resulting in enhanced thermolability and reduced AlF4/--induced adenylyl cyclase stimulation and trypsin protection, since activation by AlF4/- requires bound GDP.",
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T1 - A novel mutation in the switch 3 region of G(s)α in a patient with albright hereditary osteodystrophy impairs GDP binding and receptor activation

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N2 - Albright hereditary osteodystrophy (AHO), a disorder characterized by skeletal abnormalities and obesity, is associated with heterozygous inactivating mutations in the gene for G(s)α. A novel G(s)α mutation encoding the substitution of tryptophan for a nonconserved arginine within the switch 3 region (G(s)α R258W) was identified in an AHO patient. Although reverse transcription-polymerase chain reaction studies demonstrated that mRNA expression from wild type and mutant alleles was similar, G(s)α expression in erythrocyte membranes from the affected patient was reduced by 50%. A G(s)α R258W cDNA, as well as one with arginine replaced by alanine (G(s)α R258A), was generated, and the biochemical properties of in vitro transcription/translation products were examined. When reconstituted with cyc- membranes, both mutant proteins were able to stimulate adenylyl cyclase normally in the presence of guanosine-5'-O-(3-thiotriphosphate) (GTPγS) but had decreased ability in the presence of isoproterenol or AlF4 (a mixture of 10 μM AlCl3 and 10 mM NaF). The ability of each mutant to bind and be activated by GTPγS or AlF4/- was assessed by trypsin protection assays. Both mutants were protected normally by GTPγS but showed reduced protection in the presence of AlF4/-. The addition of excess GDP (2 mM) was able to rescue the ability of AlF4/- to protect the mutants, suggesting that they might have reduced affinity for GDP. A G(s)α R258A mutant purified from Escherichia coli had decreased affinity for GDP and an apparent rate of GDP release that was 10-fold greater than that of wild type G(s)α. Sucrose density gradient analysis demonstrated that both G(s)α R258W and G(s)α R258A were thermolabile at higher temperatures and that denaturation of both mutants was prevented by the presence of 0.1 mM GTPγS or 2 mM GDP. The crystal structure of G(s)α demonstrates that Arg258 interacts with a conserved residue in the helical domain (Gln170). Arg258 substitutions would be predicted to open the cleft between the GTPase and helical domains, allowing for increased GDP release in the inactive state, resulting in enhanced thermolability and reduced AlF4/--induced adenylyl cyclase stimulation and trypsin protection, since activation by AlF4/- requires bound GDP.

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