Abstract
Agonists for Gi-coupled receptors augment Gs-stimulated cAMP synthesis in human embryonic kidney (HEK) 293 cells transiently expressing the type II isozyme of adenylylcyclase (AC-II). This augmentation, mediated by beta gamma subunits released from activated Gi, can be blocked by expression of the alpha subunit (alpha t) of retinal transducin (Gt), which presumably sequesters free beta gamma (Federman, A. D., Conklin, B. R., Schrader, K. A., Reed, R. R., and Bourne, H. R. (1992) Nature 356, 159-161). The alpha subunit of Gq, representing a G protein family distinct from both Gs and Gi, mimicked the inhibitory effect of alpha t, suggesting that hormonal stimulation of endogenous Gq might also release beta gamma subunits and thereby augment AC-II activity. Agonists for either of two Gq-coupled receptors did augment Gs-stimulated cAMP synthesis in HEK-293 cells expressing AC-II, but this effect was not blocked by expression of alpha t. The increased stimulation of AC-II was probably not mediated by the release of beta gamma subunits from Gq but rather by activation of protein kinase C (PKC) because of the following. (a) Phorbol esters, which activate PKC directly, elevated cAMP 2-fold in HEK-293 cells transfected with AC-II; this increase was synergistic with Gs-mediated activation of AC-II. (b) Treatments that partially inhibit or down-regulate PKC also partially prevented stimulation of AC-II by phorbol esters or by agonists for Gq-coupled receptors. Taken together, these results indicate that AC-II can integrate regulatory signals transmitted by at least three classes of G proteins; extracellular signals acting through Gs are enhanced synergistically by simultaneous signals transduced by Gi or Gq and mediated via beta gamma or PKC, respectively.
Highlights
From the $Cell Biology Program, Departments of Pharmacologyand Medicine and Cardiovascular Research Institute, University of California School of Medicine, San Francisco, California 94143 and the §Department of Phrmmology, University of Texas Southwestern Medical Center, Dallas, Texas 75235
protein kinaseC (PKC) directly, elevatedcAMP 2-fold in human embryonic kidney (HEK)-293cells support the hypothesis [3, 5] that AC-I1 acts as a molecular transfected with AC-11; this increase was synergistic coincidence detector, responding to simultaneous signals with G.-mediated activation of AC-11. ( b ) Treatments that partially inhibitor down-regulate PKC partially prevented stimulationof AC-I1 by phorbol esters or by agonists for G,coupled receptors. These results indicate that AC-I1 can integrate regulatory signals transmittedby at least three classes of G proteins; extracellular signals acting throughG. are enhanced synergistically by simultaneous signals transduced by Gi or G, and mediated via By or PKC, transmitted by G. (a,.GTP) andGi (By)
AC-I1 was already known to respond to coincident signals from two other G proteins, GBand Gi [3, 5]
Summary
Receptors and rho* suggests that it can bind and sequester a species of ,&y subunit that is capable of stimulating AC-11. In the presence ofAC-11, carbachol (an mlR agonist) caused a 2-fold increase in CAMP; this increase was augmented synergistically by co-transfection with a.-Q227L(Fig. 3). Agonist stimulation of a transfected bombesin receptor (BomR), which is thought to couple to G,-like proteins, produced a similar profile of cAMP responses (not shown). By one or more proteins in the G, family and that it results in synthesis of inositol trisphosphate and diacylglycerol,second messengers that mobilize intracellular Ca" and activate PKC, respectively To test whether these second messengers account for G,dependentstimulation of AC-I1 inHEK-293 cells, we bypassed G, by directly elevating Ca2+with ionomycin, a calcium ionophore [32], or by directly stimulating PKC with phorbol 12,13-dibutyrate (PdBu), which mimics diacylglycerol [33]. After exposure to 500 nM PdBu for 24 h, cAMP was elevated by -20% in HEK293 cells expressing AC-11, a,-Q227L, and mlR; this 24-h treatment reduced by -50% the increase in cellular cAMP elicited by subsequentadministration of PdBu or by carbachol, an mlRagonist (Fig. 6).
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