Interaction of G(sα) with the cytosolic domains of mammalian adenylyl cyclase

Roger K. Sunahara, Carmen W. Dessauer, Richard E. Whisnant, Christiane Kleuss, Alfred G. Gilman

Research output: Contribution to journalArticle

152 Citations (Scopus)

Abstract

Forskolin- and G(sα)-stimulated adenylyl cyclase activity is observed after mixture of two independently-synthesized ~25-kDa cytosolic fragments derived from mammalian adenylyl cyclases (native M(r) ~ 120,000). The C(1a) domain from type V adenylyl cyclase (VC1) and the C2 domain from type II adenylyl cyclase (IIC2) can both be expressed in large quantities and purified to homogeneity. When mixed, their maximally stimulated specific activity, 150 μmol/min/mg protein, substantially exceeds values observed previously with the intact enzyme. A soluble, high-affinity complex containing one molecule each of VC1, IIC2, and guanosine 5'-O-(3-thio- triphosphate) (GTPγS)-G(sα) is responsible for the observed enzymatic activity and can be isolated. In addition, GTPγS-G(sα) interacts with homodimers of IIC2 to form a heterodimeric complex (one molecule each of G(sα) and IIC2) but not detectably with homodimers of VC1. Nevertheless, G(sα) can be cross-linked to VC1 in the activated heterotrimeric complex of VC1, IIC2, and G(sα), indicating its proximity to both components of the enzyme that are required for efficient catalysis. These results and those in the accompanying report (Dessauer, C. W., Scully, T. T., and Gilman, A. G. (1997) J. Biol. Chem. 272, 22272-22277) suggest that activators of adenylyl cyclase facilitate formation of a single, high-activity catalytic site at the interface between C1 and C2.

Original languageEnglish (US)
Pages (from-to)22265-22271
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number35
DOIs
StatePublished - Aug 29 1997

Fingerprint

Adenylyl Cyclases
Guanosine 5'-O-(3-Thiotriphosphate)
Molecules
Colforsin
Enzymes
Catalysis
Catalyst activity
Catalytic Domain
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Sunahara, R. K., Dessauer, C. W., Whisnant, R. E., Kleuss, C., & Gilman, A. G. (1997). Interaction of G(sα) with the cytosolic domains of mammalian adenylyl cyclase. Journal of Biological Chemistry, 272(35), 22265-22271. https://doi.org/10.1074/jbc.272.35.22265

Interaction of G(sα) with the cytosolic domains of mammalian adenylyl cyclase. / Sunahara, Roger K.; Dessauer, Carmen W.; Whisnant, Richard E.; Kleuss, Christiane; Gilman, Alfred G.

In: Journal of Biological Chemistry, Vol. 272, No. 35, 29.08.1997, p. 22265-22271.

Research output: Contribution to journalArticle

Sunahara, RK, Dessauer, CW, Whisnant, RE, Kleuss, C & Gilman, AG 1997, 'Interaction of G(sα) with the cytosolic domains of mammalian adenylyl cyclase', Journal of Biological Chemistry, vol. 272, no. 35, pp. 22265-22271. https://doi.org/10.1074/jbc.272.35.22265
Sunahara, Roger K. ; Dessauer, Carmen W. ; Whisnant, Richard E. ; Kleuss, Christiane ; Gilman, Alfred G. / Interaction of G(sα) with the cytosolic domains of mammalian adenylyl cyclase. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 35. pp. 22265-22271.
@article{49f45aeee37943028dbb18818c27e6d2,
title = "Interaction of G(sα) with the cytosolic domains of mammalian adenylyl cyclase",
abstract = "Forskolin- and G(sα)-stimulated adenylyl cyclase activity is observed after mixture of two independently-synthesized ~25-kDa cytosolic fragments derived from mammalian adenylyl cyclases (native M(r) ~ 120,000). The C(1a) domain from type V adenylyl cyclase (VC1) and the C2 domain from type II adenylyl cyclase (IIC2) can both be expressed in large quantities and purified to homogeneity. When mixed, their maximally stimulated specific activity, 150 μmol/min/mg protein, substantially exceeds values observed previously with the intact enzyme. A soluble, high-affinity complex containing one molecule each of VC1, IIC2, and guanosine 5'-O-(3-thio- triphosphate) (GTPγS)-G(sα) is responsible for the observed enzymatic activity and can be isolated. In addition, GTPγS-G(sα) interacts with homodimers of IIC2 to form a heterodimeric complex (one molecule each of G(sα) and IIC2) but not detectably with homodimers of VC1. Nevertheless, G(sα) can be cross-linked to VC1 in the activated heterotrimeric complex of VC1, IIC2, and G(sα), indicating its proximity to both components of the enzyme that are required for efficient catalysis. These results and those in the accompanying report (Dessauer, C. W., Scully, T. T., and Gilman, A. G. (1997) J. Biol. Chem. 272, 22272-22277) suggest that activators of adenylyl cyclase facilitate formation of a single, high-activity catalytic site at the interface between C1 and C2.",
author = "Sunahara, {Roger K.} and Dessauer, {Carmen W.} and Whisnant, {Richard E.} and Christiane Kleuss and Gilman, {Alfred G.}",
year = "1997",
month = "8",
day = "29",
doi = "10.1074/jbc.272.35.22265",
language = "English (US)",
volume = "272",
pages = "22265--22271",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "35",

}

TY - JOUR

T1 - Interaction of G(sα) with the cytosolic domains of mammalian adenylyl cyclase

AU - Sunahara, Roger K.

AU - Dessauer, Carmen W.

AU - Whisnant, Richard E.

AU - Kleuss, Christiane

AU - Gilman, Alfred G.

PY - 1997/8/29

Y1 - 1997/8/29

N2 - Forskolin- and G(sα)-stimulated adenylyl cyclase activity is observed after mixture of two independently-synthesized ~25-kDa cytosolic fragments derived from mammalian adenylyl cyclases (native M(r) ~ 120,000). The C(1a) domain from type V adenylyl cyclase (VC1) and the C2 domain from type II adenylyl cyclase (IIC2) can both be expressed in large quantities and purified to homogeneity. When mixed, their maximally stimulated specific activity, 150 μmol/min/mg protein, substantially exceeds values observed previously with the intact enzyme. A soluble, high-affinity complex containing one molecule each of VC1, IIC2, and guanosine 5'-O-(3-thio- triphosphate) (GTPγS)-G(sα) is responsible for the observed enzymatic activity and can be isolated. In addition, GTPγS-G(sα) interacts with homodimers of IIC2 to form a heterodimeric complex (one molecule each of G(sα) and IIC2) but not detectably with homodimers of VC1. Nevertheless, G(sα) can be cross-linked to VC1 in the activated heterotrimeric complex of VC1, IIC2, and G(sα), indicating its proximity to both components of the enzyme that are required for efficient catalysis. These results and those in the accompanying report (Dessauer, C. W., Scully, T. T., and Gilman, A. G. (1997) J. Biol. Chem. 272, 22272-22277) suggest that activators of adenylyl cyclase facilitate formation of a single, high-activity catalytic site at the interface between C1 and C2.

AB - Forskolin- and G(sα)-stimulated adenylyl cyclase activity is observed after mixture of two independently-synthesized ~25-kDa cytosolic fragments derived from mammalian adenylyl cyclases (native M(r) ~ 120,000). The C(1a) domain from type V adenylyl cyclase (VC1) and the C2 domain from type II adenylyl cyclase (IIC2) can both be expressed in large quantities and purified to homogeneity. When mixed, their maximally stimulated specific activity, 150 μmol/min/mg protein, substantially exceeds values observed previously with the intact enzyme. A soluble, high-affinity complex containing one molecule each of VC1, IIC2, and guanosine 5'-O-(3-thio- triphosphate) (GTPγS)-G(sα) is responsible for the observed enzymatic activity and can be isolated. In addition, GTPγS-G(sα) interacts with homodimers of IIC2 to form a heterodimeric complex (one molecule each of G(sα) and IIC2) but not detectably with homodimers of VC1. Nevertheless, G(sα) can be cross-linked to VC1 in the activated heterotrimeric complex of VC1, IIC2, and G(sα), indicating its proximity to both components of the enzyme that are required for efficient catalysis. These results and those in the accompanying report (Dessauer, C. W., Scully, T. T., and Gilman, A. G. (1997) J. Biol. Chem. 272, 22272-22277) suggest that activators of adenylyl cyclase facilitate formation of a single, high-activity catalytic site at the interface between C1 and C2.

UR - http://www.scopus.com/inward/record.url?scp=0030612427&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030612427&partnerID=8YFLogxK

U2 - 10.1074/jbc.272.35.22265

DO - 10.1074/jbc.272.35.22265

M3 - Article

C2 - 9268375

AN - SCOPUS:0030612427

VL - 272

SP - 22265

EP - 22271

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 35

ER -