A Region of Adenylyl Cyclase 2 Critical for Regulation by G Protein βγ Subunits

Chen, Jianqiang; DeVivo, Michael J.; Dingus, Jane; Harry, Anya; Li, Jingrong; Sui, Jinliang; Carty, D. J.; Blank, Jonathan L.; Exton, John H.; Stoffel, Robert H.; Inglese, James; Lefkowitz, Robert J.; Logothetis, Diomedes E.; Hildebrandt, John D.; lyengar, Ravi

doi:10.1126/science.7761832

Cited by 260 publications

(191 citation statements)

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“…Noticeably, it was recently demonstrated that fl?' subunits of G proteins could bind to a QXXER motif present on several effectors such as adenylyl cyclase 2 and muscarinic potassium channels [28]. This motif is also present in the AID sequence of three neuronal ~ subunits (classes A, B and E) where the arginine residue represents a non-conserved AID amino acid which is absent in the remaining (z~ subunits (classes S~ C and D).…”

Section: Discussionmentioning

confidence: 99%

Identification of critical amino acids involved in α₁‐β interaction in voltage‐dependent Ca²⁺ channels

et al. 1996

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In voltage-dependent Ca 2+ channels, the al and subunits interact via two cytoplasmic regions defined as the Alpha Interaction Domain (AID) and Beta Interaction Domain (BID). Several novel amino acids for that interaction have now been mapped in both domains by point mutations. It was found that three of the nine amino acids in AID and four of the eight BID amino acids tested were essential for the interaction. Whereas the important AID amino acids were clustered around five residues, the important BID residues were more widely distributed within a larger 16 amino acid sequence. The affinity of the AID A GST fusion protein for the four interacting/]lb BID mutants was not significantly altered compared with the wild-type ~b despite the close localization of mutated residues to disruptive BID amino acids. Expression of these interactive S] mutants with the fulllength OLIA subunit only slightly modified the stimulation efficiency when compared with the wild-type ~b subunit. Our data suggest that non-disruptive BID sequence alterations do not dramatically affect the/] subunit-induced current stimulation.

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Section: Discussionmentioning

confidence: 99%

Identification of critical amino acids involved in α₁‐β interaction in voltage‐dependent Ca²⁺ channels

et al. 1996

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“…AC2 assays have been described (6). When required the peptides were mixed with adenylyl cyclase containing membranes and held on ice for 10 min prior to assays.…”

Section: Methodsmentioning

confidence: 99%

“…AC2 was expressed in Sf9 cells by infection with recombinant baculovirus (10). AC2 assays have been described (6). Bovine AC1 (11) was epitope tagged at the N terminus with the FLAG epitope (10) and expressed in Sf9 cells by baculovirus infection.…”

Section: Methodsmentioning

confidence: 99%

“…Regions of G␤␥ complex involved in communicating the signal to effectors have not been well characterized. We had identified the region of residues 956-982 of adenylyl cyclase (AC) 2 as being involved in receiving signals from G␤␥ (6). By using the yeast twohybrid system, the AC2 region of residues 956-982 has been subsequently shown to interact with G␤ but not G␥ subunits (7).…”

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confidence: 99%

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A surface on the G protein β-subunit involved in interactions with adenylyl cyclases

Chen¹,

Weng²,

Li³

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Receptor activation of heterotrimeric G proteins dissociates G␣ from the G␤␥ complex, allowing both to regulate effectors. Little is known about the effectorinteraction regions of G␤␥. We had used molecular modeling to dock a peptide encoding the region of residues 956-982 of adenylyl cyclase (AC) 2 onto G␤ to identify residues on G␤ that may interact with effectors. Based on predictions from the model, we synthesized peptides encoding sequences of residues 86-105 (G␤86-105) and 115-135 (G␤115-135) from G␤. The G␤86-105 peptide inhibited G␤␥ stimulation of AC2 and blocked G␤␥ inhibition of AC1 and by itself inhibited calmodulin-stimulated AC1, thus displaying partial agonist activity. Substitution of Met-101 with Asn in this peptide resulted in the loss of both the inhibitory and partial agonist activities. Most activities of the G␤115-135 peptide were similar to those of G␤86-105 but G␤115-135 was less efficacious in blocking G␤␥ inhibition of AC1. Substitution of Tyr-124 with Val in the G␤115-135 peptide diminished all of its activities. These results identify the region encoded by amino acids 84-143 of G␤ as a surface that is involved in transmitting signals to effectors.Heterotrimeric G proteins serve as signal transducers for a wide variety of receptors. Both G␣ and G␤␥ subunits can communicate receptor signals (1-5). Regions of G␤␥ complex involved in communicating the signal to effectors have not been well characterized. We had identified the region of residues 956-982 of adenylyl cyclase (AC) 2 as being involved in receiving signals from G␤␥ (6). By using the yeast twohybrid system, the AC2 region of residues 956-982 has been subsequently shown to interact with G␤ but not G␥ subunits (7). In recent studies we found that the peptide encoding residues 956-982 of AC2 can be crosslinked to G␤ when it is part of the free G␤␥ complex but not when it is part of the heterotrimer, indicating that the putative binding surface on G␤ for the AC2 peptide is occluded by interactions with G␣. On the basis of constraints deduced from the crosslinking studies and other biophysical criteria, we docked the AC2 peptide containing residues 956-982 onto the crystal structure of G␤ by using molecular modeling techniques (8). From this docking model, we have identified the regions of G␤ that are predicted to interact with the AC2 peptide. Herein we have tested whether peptides encoding the effector-interaction surface of G␤ predicted from the modeling (8) can modulate G␤␥ regulation of AC1 and AC2. MATERIALS AND METHODSMaterials. Reagents for peptide synthesis were from Bachem. [␣-32 P]ATP was from New England Nuclear. Tissue culture reagents and fetal calf serum was from GIBCO. All other chemicals used were the highest grade available.Peptide Synthesis. Peptides were synthesized on an Applied Biosystems peptide synthesizer (model 431A) and purified by HPLC on acetonitrile gradients. Purified peptides were lyophilized and stored at Ϫ20ЊC. When required peptides were dissolved in water to a final concentration of 1-3 mM....

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“…A consensus sequence (QXXER) has been proposed to be involved in the interaction of G␤␥ subunits with adenylyl cyclase type 2, inwardly rectifying K ϩ channels, and phospholipase C␤ (15). A related consensus sequence is within or adjacent to the region of the COOH terminus of inwardly rectifying K ϩ channels which is implicated in binding and regulation by G␤␥ subunits (16,17).…”

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confidence: 99%

Molecular determinants of inactivation and G protein modulation in the intracellular loop connecting domains I and II of the calcium channel α _1A subunit

Herlitze

Hockerman

Scheuer

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

193

176

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Synaptic transmission is regulated by G protein-coupled receptors whose activation releases G protein ␤␥ subunits that modulate presynaptic Ca 2؉ channels. The sequence motif QXXER has been proposed to be involved in the interaction between G protein ␤␥ subunits and target proteins including adenylyl cyclase 2. This motif is present in the intracellular loop connecting domains I and II (L I-II ) of Ca 2؉ channel ␣ 1A subunits, which are modulated by G proteins, but not in ␣ 1C subunits, which are not modulated. Peptides containing the QXXER motif from adenylate cyclase 2 or from ␣ 1A block G protein modulation but a mutant peptide containing the sequence AXXAA does not, suggesting that the QXXER-containing peptide from ␣ 1A can competitively inhibit G␤␥ modulation. Conversion of the R in the QQIER sequence of ␣ 1A to E as in ␣ 1C slows channel inactivation and shifts the voltage dependence of steady-state inactivation to more positive membrane potentials. Conversion of the final E in the QQLEE sequence of ␣ 1C to R has opposite effects on voltage-dependent inactivation, although the changes are not as large as those for ␣ 1A . Mutation of the QQIER sequence in ␣ 1A to QQIEE enhanced G protein modulation, and mutation to QQLEE as in ␣ 1C greatly reduced G protein modulation and increased the rate of reversal of G protein effects. These results indicate that the QXXER motif in L I-II is an important determinant of both voltage-dependent inactivation and G protein modulation, and that the amino acid in the third position of this motif has an unexpectedly large inf luence on modulation by G␤␥. Overlap of this motif with the consensus sequence for binding of Ca 2؉ channel ␤ subunits suggests that this region of L I-II is important for three different modulatory inf luences on Ca 2؉ channel activity.Neuronal voltage-gated Ca 2ϩ channels are involved in multiple cellular functions including neurotranstransmitter release, Ca 2ϩ -mediated regulatory processes, and generation of dendritic action potentials. They consist of complexes of a poreforming ␣ 1 subunit of 190-250 kDa in association with ␣ 2 ␦ and ␤ subunits (1, 2). Electrophysiological and pharmacological studies distinguish at least six classes of Ca 2ϩ channel currents designated L-, N-, P-, Q-, R-, and T-type (2, 3). Ca 2ϩ channels containing ␣ 1C or ␣ 1D subunits are thought to be responsible for L-type currents, ␣ 1B for N-type currents, and ␣ 1A for both P-and Q-type calcium currents (2, 4). A major goal of current research is to correlate the observed differences among the properties of these channel types with the structures of their ␣ 1 subunits.P͞Q-type and N-type Ca 2ϩ channels containing ␣ 1A or ␣ 1B subunits differ from L-type Ca 2ϩ channels containing ␣ 1C or ␣ 1D subunits in voltage-dependent inactivation (5-8) and in modulation by G proteins (9). Voltage-dependent inactivation of L-type Ca 2ϩ channels containing cloned ␣ 1C is slower than inactivation of P͞Q-type Ca 2ϩ channels containing cloned ␣ 1A expressed with the same auxiliary subun...

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A Region of Adenylyl Cyclase 2 Critical for Regulation by G Protein βγ Subunits

Cited by 260 publications

References 33 publications

Identification of critical amino acids involved in α₁‐β interaction in voltage‐dependent Ca²⁺ channels

Identification of critical amino acids involved in α₁‐β interaction in voltage‐dependent Ca²⁺ channels

A surface on the G protein β-subunit involved in interactions with adenylyl cyclases

Molecular determinants of inactivation and G protein modulation in the intracellular loop connecting domains I and II of the calcium channel α _1A subunit

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A Region of Adenylyl Cyclase 2 Critical for Regulation by G Protein βγ Subunits

Cited by 260 publications

References 33 publications

Identification of critical amino acids involved in α1‐β interaction in voltage‐dependent Ca2+ channels

Identification of critical amino acids involved in α1‐β interaction in voltage‐dependent Ca2+ channels

A surface on the G protein β-subunit involved in interactions with adenylyl cyclases

Molecular determinants of inactivation and G protein modulation in the intracellular loop connecting domains I and II of the calcium channel α 1A subunit

Contact Info

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Identification of critical amino acids involved in α₁‐β interaction in voltage‐dependent Ca²⁺ channels

Identification of critical amino acids involved in α₁‐β interaction in voltage‐dependent Ca²⁺ channels

Molecular determinants of inactivation and G protein modulation in the intracellular loop connecting domains I and II of the calcium channel α _1A subunit