Discovery of LRE1 as a specific and allosteric inhibitor of soluble adenylyl cyclase

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Abstract

The prototypical second messenger cAMP regulates a wide variety of physiological processes. It can simultaneously mediate diverse functions by acting locally within independently-regulated microdomains. In mammalian cells, two types of adenylyl cyclase generate cAMP; G protein regulated transmembrane adenylyl cyclases and bicarbonate- calcium- and ATP-regulated soluble adenylyl cyclase (sAC). Because each type of cyclase regulates distinct microdomains, understanding cAMP signaling demands methods to distinguish between them. We developed a mass spectrometry based adenylyl cyclase assay which we used to identify a novel sAC-specific inhibitor, LRE1. LRE1 binds to the bicarbonate activator binding site and inhibits sAC via a unique allosteric mechanism. LRE1 prevents sAC-dependent processes in cellular and physiological systems and facilitates exploration of the therapeutic potential of sAC inhibition.

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Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor.

Y. Chen, M Cann, T N Litvin … (2000)

Spermatozoa undergo a poorly understood activation process induced by bicarbonate and mediated by cyclic adenosine 3',5'-monophosphate (cAMP). It has been assumed that bicarbonate mediates its effects through changes in intracellular pH or membrane potential; however, we demonstrate here that bicarbonate directly stimulates mammalian soluble adenylyl cyclase (sAC) activity in vivo and in vitro in a pH-independent manner. sAC is most similar to adenylyl cyclases from cyanobacteria, and bicarbonate regulation of cyclase activity is conserved in these early forms of life. sAC is also expressed in other bicarbonate-responsive tissues, which suggests that bicarbonate regulation of cAMP signaling plays a fundamental role in many biological systems.

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Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells.

Gaelle Chaffard, Blanca Rubi, Pierre Maechler … (2004)

Rat insulinoma-derived INS-1 cells constitute a widely used beta-cell surrogate. However, due to their nonclonal nature, INS-1 cells are heterogeneous and are not stable over extended culture periods. We have isolated clonal INS-1E cells from parental INS-1 based on both their insulin content and their secretory responses to glucose. Here we describe the stable differentiated INS-1E beta-cell phenotype over 116 passages (no. 27-142) representing a 2.2-yr continuous follow-up. INS-1E cells can be safely cultured and used within passages 40-100 with average insulin contents of 2.30 +/- 0.11 microg/million cells. Glucose-induced insulin secretion was dose-related and similar to rat islet responses. Secretion saturated with a 6.2-fold increase at 15 mm glucose, showing a 50% effective concentration of 10.4 mm. Secretory responses to amino acids and sulfonylurea were similar to those of islets. Moreover, INS-1E cells retained the amplifying pathway, as judged by glucose-evoked augmentation of insulin release in a depolarized state. Regarding metabolic parameters, INS-1E cells exhibited glucose dose-dependent elevations of NAD(P)H, cytosolic Ca(2+), and mitochondrial Ca(2+) levels. In contrast, mitochondrial membrane potential, ATP levels, and cell membrane potential were all fully activated by 7.5 mm glucose. Using the perforated patch clamp technique, 7.5 and 15 mm glucose elicited electrical activity to a similar degree. A K(ATP) current was identified in whole cell voltage clamp using diazoxide and tolbutamide. As in native beta-cells, tolbutamide induced electrical activity, indicating that the K(ATP)conductance is important in setting the resting potential. Therefore, INS-1E cells represent a stable and valuable beta-cell model.

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Cyclic AMP produced inside mitochondria regulates oxidative phosphorylation.

Rebeca Acín-Pérez, Eric Salazar, Margarita Kamenetsky … (2009)

Mitochondria constantly respond to changes in substrate availability and energy utilization to maintain cellular ATP supplies, and at the same time control reactive oxygen radical (ROS) production. Reversible phosphorylation of mitochondrial proteins has been proposed to play a fundamental role in metabolic homeostasis, but very little is known about the signaling pathways involved. We show here that protein kinase A (PKA) regulates ATP production by phosphorylation of mitochondrial proteins, including subunits of cytochrome c oxidase. The cyclic AMP (cAMP), which activates mitochondrial PKA, does not originate from cytoplasmic sources but is generated within mitochondria by the carbon dioxide/bicarbonate-regulated soluble adenylyl cyclase (sAC) in response to metabolically generated carbon dioxide. We demonstrate for the first time the existence of a CO(2)-HCO(3)(-)-sAC-cAMP-PKA (mito-sAC) signaling cascade wholly contained within mitochondria, which serves as a metabolic sensor modulating ATP generation and ROS production in response to nutrient availability.

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Author and article information

Journal

Journal ID (nlm-journal-id): 101231976

Journal ID (pubmed-jr-id): 32624

Journal ID (nlm-ta): Nat Chem Biol

Journal ID (iso-abbrev): Nat. Chem. Biol.

Title: Nature chemical biology

ISSN (Print): 1552-4450

ISSN (Electronic): 1552-4469

Publication date Nihms-submitted: 27 May 2016

Publication date (Electronic): 22 August 2016

Publication date (Print): October 2016

Publication date PMC-release: 01 April 2017

Volume: 12

Issue: 10

Pages: 838-844

Affiliations

[1 ]Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA

[2 ]Department of Biochemistry, University of Bayreuth, 95440 Bayreuth, Germany

[3 ]Department of Veterinary and Animal Science, University of Massachusetts, Amherst MA, USA

[4 ]Brain and Mind Research Institute, Weill Cornell Medical College, New York, NY 10065, USA

[5 ]The High-Throughput Screening and Spectroscopy Resource Center, The Rockefeller University, New York, NY 10065, USA

[6 ]Department of Dermatology, Weill Cornell Medical College, New York, NY 10065, USA

[7 ]Helmholtz-Zentrum fur Infektionsforschung, 38124 Braunschweig, Germany

Article

Manuscript ID: NIHMS789743

DOI: 10.1038/nchembio.2151

PMC ID: 5030147

PubMed ID: 27547922

SO-VID: 472dfc3c-c2da-4901-b3dc-49cb2f62a573

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Discovery of LRE1 as a specific and allosteric inhibitor of soluble adenylyl cyclase

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Most cited references 53

Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor.

Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells.

Cyclic AMP produced inside mitochondria regulates oxidative phosphorylation.

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