Mutation of the highly conserved Arg165 and Glu168 residues of human Gsα disrupts the αD-αE loop and enhances basal GDP/GTP exchange rate

María Victoria Hinrichs, Martin Montecino, Marta Bunster, Juan Olate

Resultado de la investigación: Article

1 Cita (Scopus)

Resumen

G protein signalling regulates a wide range of cellular processes such as motility, differentiation, secretion, neurotransmission, and cell division. G proteins consist of three subunits organized as a Gα monomer associated with a Gβγ heterodimer. Structural studies have shown that Gα subunits are constituted by two domains: a Ras-like domain, also called the GTPase domain (GTPaseD), and an helical domain (HD), which is unique to heterotrimeric G-proteins. The HD display significantly higher primary structure diversity than the GTPaseD. Regardless of this diversity, there are small regions of the HD which show high degree of identity with residues that are 100% conserved. One of such regions is the α helixD-α helixE loop (αD-αE) in the HD, which contains the consensus aminoacid sequence R*-[RSA]-[RSAN]-E*-[YF]-[QH]-L in all mammalian Gα subunits. Interestingly, the highly conserved arginine (R*) and glutamic acid (E*) residues form a salt bridge that stabilizes the αD-αE loop, that is localized in the top of the cleft formed between the GTPaseD and HD. Because the guanine nucleotide binding site is deeply buried in this cleft and those interdomain interactions are playing an important role in regulating the basal GDP/GTP nucleotide exchange rate of Gα subunits, we studied the role of these highly conserved R and E residues in Gα function. In the present study, we mutated the human Gsα R165 and E 168 residues to alanine (A), thus generating the R165 → A, E168 → A, and R165/E168 → A mutants. We expressed these human Gsα (hGsα) mutants in bacteria as histidine tagged proteins, purified them by niquel-agarose chromatography and studied their nucleotide exchange properties. We show that the double R 165/E168 → A mutant exhibited a fivefold increased GTP binding kinetics, a higher GDP dissociation rate, and an augmented capacity to activate adenylyl cyclase. Structure analysis showed that disruption of the salt bridge between R165 and E168 by the introduced mutations, caused important structural changes in the HD at the αD-αE loop (residues 160-175) and in the GTPaseD at a region required for Gsα activation by the receptor (residues 308-315). In addition, other two GTPaseD regions that surround the GTP binding site were also affected.

Idioma originalEnglish
Páginas (desde-hasta)409-417
Número de páginas9
PublicaciónJournal of Cellular Biochemistry
Volumen93
N.º2
DOI
EstadoPublished - 2004

Huella dactilar

GTP Phosphohydrolases
Guanosine Triphosphate
Mutation
GTP-Binding Proteins
Nucleotides
Salts
Binding Sites
Heterotrimeric GTP-Binding Proteins
Agarose Chromatography
Guanine Nucleotides
Consensus Sequence
Chromatography
Histidine
Adenylyl Cyclases
Synaptic Transmission
Alanine
Cell Division
Sepharose
Arginine
Glutamic Acid

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Citar esto

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title = "Mutation of the highly conserved Arg165 and Glu168 residues of human Gsα disrupts the αD-αE loop and enhances basal GDP/GTP exchange rate",
abstract = "G protein signalling regulates a wide range of cellular processes such as motility, differentiation, secretion, neurotransmission, and cell division. G proteins consist of three subunits organized as a Gα monomer associated with a Gβγ heterodimer. Structural studies have shown that Gα subunits are constituted by two domains: a Ras-like domain, also called the GTPase domain (GTPaseD), and an helical domain (HD), which is unique to heterotrimeric G-proteins. The HD display significantly higher primary structure diversity than the GTPaseD. Regardless of this diversity, there are small regions of the HD which show high degree of identity with residues that are 100{\%} conserved. One of such regions is the α helixD-α helixE loop (αD-αE) in the HD, which contains the consensus aminoacid sequence R*-[RSA]-[RSAN]-E*-[YF]-[QH]-L in all mammalian Gα subunits. Interestingly, the highly conserved arginine (R*) and glutamic acid (E*) residues form a salt bridge that stabilizes the αD-αE loop, that is localized in the top of the cleft formed between the GTPaseD and HD. Because the guanine nucleotide binding site is deeply buried in this cleft and those interdomain interactions are playing an important role in regulating the basal GDP/GTP nucleotide exchange rate of Gα subunits, we studied the role of these highly conserved R and E residues in Gα function. In the present study, we mutated the human Gsα R165 and E 168 residues to alanine (A), thus generating the R165 → A, E168 → A, and R165/E168 → A mutants. We expressed these human Gsα (hGsα) mutants in bacteria as histidine tagged proteins, purified them by niquel-agarose chromatography and studied their nucleotide exchange properties. We show that the double R 165/E168 → A mutant exhibited a fivefold increased GTP binding kinetics, a higher GDP dissociation rate, and an augmented capacity to activate adenylyl cyclase. Structure analysis showed that disruption of the salt bridge between R165 and E168 by the introduced mutations, caused important structural changes in the HD at the αD-αE loop (residues 160-175) and in the GTPaseD at a region required for Gsα activation by the receptor (residues 308-315). In addition, other two GTPaseD regions that surround the GTP binding site were also affected.",
keywords = "Adenylyl cyclase, G-protein, Gsα",
author = "Hinrichs, {Mar{\'i}a Victoria} and Martin Montecino and Marta Bunster and Juan Olate",
year = "2004",
doi = "10.1002/jcb.20193",
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journal = "Journal of Cellular Biochemistry",
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Mutation of the highly conserved Arg165 and Glu168 residues of human Gsα disrupts the αD-αE loop and enhances basal GDP/GTP exchange rate. / Hinrichs, María Victoria; Montecino, Martin; Bunster, Marta; Olate, Juan.

En: Journal of Cellular Biochemistry, Vol. 93, N.º 2, 2004, p. 409-417.

Resultado de la investigación: Article

TY - JOUR

T1 - Mutation of the highly conserved Arg165 and Glu168 residues of human Gsα disrupts the αD-αE loop and enhances basal GDP/GTP exchange rate

AU - Hinrichs, María Victoria

AU - Montecino, Martin

AU - Bunster, Marta

AU - Olate, Juan

PY - 2004

Y1 - 2004

N2 - G protein signalling regulates a wide range of cellular processes such as motility, differentiation, secretion, neurotransmission, and cell division. G proteins consist of three subunits organized as a Gα monomer associated with a Gβγ heterodimer. Structural studies have shown that Gα subunits are constituted by two domains: a Ras-like domain, also called the GTPase domain (GTPaseD), and an helical domain (HD), which is unique to heterotrimeric G-proteins. The HD display significantly higher primary structure diversity than the GTPaseD. Regardless of this diversity, there are small regions of the HD which show high degree of identity with residues that are 100% conserved. One of such regions is the α helixD-α helixE loop (αD-αE) in the HD, which contains the consensus aminoacid sequence R*-[RSA]-[RSAN]-E*-[YF]-[QH]-L in all mammalian Gα subunits. Interestingly, the highly conserved arginine (R*) and glutamic acid (E*) residues form a salt bridge that stabilizes the αD-αE loop, that is localized in the top of the cleft formed between the GTPaseD and HD. Because the guanine nucleotide binding site is deeply buried in this cleft and those interdomain interactions are playing an important role in regulating the basal GDP/GTP nucleotide exchange rate of Gα subunits, we studied the role of these highly conserved R and E residues in Gα function. In the present study, we mutated the human Gsα R165 and E 168 residues to alanine (A), thus generating the R165 → A, E168 → A, and R165/E168 → A mutants. We expressed these human Gsα (hGsα) mutants in bacteria as histidine tagged proteins, purified them by niquel-agarose chromatography and studied their nucleotide exchange properties. We show that the double R 165/E168 → A mutant exhibited a fivefold increased GTP binding kinetics, a higher GDP dissociation rate, and an augmented capacity to activate adenylyl cyclase. Structure analysis showed that disruption of the salt bridge between R165 and E168 by the introduced mutations, caused important structural changes in the HD at the αD-αE loop (residues 160-175) and in the GTPaseD at a region required for Gsα activation by the receptor (residues 308-315). In addition, other two GTPaseD regions that surround the GTP binding site were also affected.

AB - G protein signalling regulates a wide range of cellular processes such as motility, differentiation, secretion, neurotransmission, and cell division. G proteins consist of three subunits organized as a Gα monomer associated with a Gβγ heterodimer. Structural studies have shown that Gα subunits are constituted by two domains: a Ras-like domain, also called the GTPase domain (GTPaseD), and an helical domain (HD), which is unique to heterotrimeric G-proteins. The HD display significantly higher primary structure diversity than the GTPaseD. Regardless of this diversity, there are small regions of the HD which show high degree of identity with residues that are 100% conserved. One of such regions is the α helixD-α helixE loop (αD-αE) in the HD, which contains the consensus aminoacid sequence R*-[RSA]-[RSAN]-E*-[YF]-[QH]-L in all mammalian Gα subunits. Interestingly, the highly conserved arginine (R*) and glutamic acid (E*) residues form a salt bridge that stabilizes the αD-αE loop, that is localized in the top of the cleft formed between the GTPaseD and HD. Because the guanine nucleotide binding site is deeply buried in this cleft and those interdomain interactions are playing an important role in regulating the basal GDP/GTP nucleotide exchange rate of Gα subunits, we studied the role of these highly conserved R and E residues in Gα function. In the present study, we mutated the human Gsα R165 and E 168 residues to alanine (A), thus generating the R165 → A, E168 → A, and R165/E168 → A mutants. We expressed these human Gsα (hGsα) mutants in bacteria as histidine tagged proteins, purified them by niquel-agarose chromatography and studied their nucleotide exchange properties. We show that the double R 165/E168 → A mutant exhibited a fivefold increased GTP binding kinetics, a higher GDP dissociation rate, and an augmented capacity to activate adenylyl cyclase. Structure analysis showed that disruption of the salt bridge between R165 and E168 by the introduced mutations, caused important structural changes in the HD at the αD-αE loop (residues 160-175) and in the GTPaseD at a region required for Gsα activation by the receptor (residues 308-315). In addition, other two GTPaseD regions that surround the GTP binding site were also affected.

KW - Adenylyl cyclase

KW - G-protein

KW - Gsα

UR - http://www.scopus.com/inward/record.url?scp=16544380270&partnerID=8YFLogxK

U2 - 10.1002/jcb.20193

DO - 10.1002/jcb.20193

M3 - Article

C2 - 15368366

AN - SCOPUS:16544380270

VL - 93

SP - 409

EP - 417

JO - Journal of Cellular Biochemistry

JF - Journal of Cellular Biochemistry

SN - 0730-2312

IS - 2

ER -