Biomedical subjects

H Ongun Onaran

Publications and source records attributed to H Ongun Onaran.

4 recordsLinked to original sources

PubMed2005

Guanine nucleotide exchange-independent activation of Gs protein by beta2-adrenoceptor.

beta2-adrenoceptor-mediated activation of Gs and adenylyl cyclase or other receptor-mediated G protein activations is believed to occur by receptor-catalyzed replacement of GDP with GTP on the alpha-subunit of the G protein. Here we showed that a beta2-adrenoceptor-Gs system, heterologously expressed in cyc- or human embryonic kidney (HEK)-293 cells, can be activated in the presence of GDP or its phosphorylation-resistant analog, guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS). The potency and maximal ability of GDP to activate Gs and adenylyl cyclase were identical to those of GTP. GDP-mediated activation of adenylyl cyclase, similar to that mediated by GTP, was concentration-dependent, required high magnesium concentrations, was inhibited by inverse agonists, and was correlated with the efficacy of receptor ligands used to stimulate the receptor. UDP did not block the GDP-mediated activation, although it completely blocked the formation of a small amount of GTP ( approximately 5% GDP) from GDP. Moreover, the activation of Gs in the presence of GDP was insensitive to cholera toxin treatment of the cells, whereas that observed in the presence of GTP was amplified by the treatment, which showed that the activation observed in the presence of GDP was not mediated by GTP. Therefore, we concluded that GDP itself could mediate beta-adrenoceptor-induced activation of Gs-adenylyl cyclase system as much as GTP. We discuss the results in the context of the current paradigm of receptor-mediated G protein activation and propose an additional mode of activation for beta2-adrenoceptor-G(s) adenylyl cyclase system where nucleotide exchange is not necessary and GDP and GTP play identical roles in receptor-induced Gs protein activation.

Adenylyl Cyclases↗

PubMed2003

Partial rescue of functional interactions of a nonpalmitoylated mutant of the G-protein G alpha s by fusion to the beta-adrenergic receptor.

Most heterotrimeric G-protein alpha subunits are posttranslationally modified by palmitoylation, a reversible process that is dynamically regulated. We analyzed the effects of Galpha(s) palmitoylation for its intracellular distribution and ability to couple to the beta-adrenergic receptor (betaAR) and stimulate adenylyl cyclase. Subcellular fractionation and immunofluorescence microscopy of stably transfected cyc(-) cells, which lack endogenous Galpha(s), showed that wild-type Galpha(s) was predominantly localized at the plasma membrane, but the mutant C3A-Galpha(s), which does not incorporate [(3)H]palmitate, was mostly associated with intracellular membranes. In agreement with this mislocalization, C3A-Galpha(s) showed neither isoproterenol- or GTPgammaS-stimulated adenylyl cyclase activation nor GTPgammaS-sensitive high-affinity agonist binding, all of which were present in the wild-type Galpha(s) expressing cells. Fusion of C3A-Galpha(s) with the betaAR [betaAR-(C3A)Galpha(s)] partially rescued its ability to induce high-affinity agonist binding and to stimulate adenylyl cyclase activity after isoproterenol or GTPgammaS treatment. In comparison to results with the WT-Galpha(s) and betaAR (betaAR-Galpha(s)) fusion protein, the betaAR-(C3A)Galpha(s) fusion protein was about half as efficient at coupling to the receptor and effector. Chemical depalmitoylation by hydroxylamine of membranes expressing betaAR-Galpha(s) reduced the high-affinity agonist binding and adenylyl cyclase activation to a similar degree as that observed in betaAR-(C3A)Galpha(s) expressing membranes. Altogether, these findings indicate that palmitoylation ensured proper localization of Galpha(s) and facilitated bimolecular interactions of Galpha(s) with the betaAR and adenylyl cyclase.

Adenylyl Cyclases↗

PubMed2002

Agonist-directed trafficking explaining the difference between response pattern of naratriptan and sumatriptan in rabbit common carotid artery.

1. Sumatriptan or eletriptan produced vasocontraction in common carotid artery (CCA) by stimulating 5HT(1B) receptors (see also Akin & Gurdal, this issue). 2. Naratriptan as a 5HT(1B/D) agonist, was unable to produce vasocontraction in this artery, but inhibited the vasocontractile response induced by sumatriptan or eletriptan. 3. All these agonists inhibited forskolin-stimulated cyclic AMP production with comparable potencies and maximal responses. This inhibition was mediated by 5HT(1B) receptors: 5HT(1B) antagonist SB216641 (1 microM) completeley antagonized sumatriptan-, eletriptan- or naratriptan-induced cyclic AMP inhibition, but 5HT(1D) antagonist BRL15572 (1 microM) did not affect this response. 4. Naratriptan-induced stimulation of 5-HT(1B) receptors resulted only in adenylate cyclase inhibition, whereas stimulation of these receptors by sumatriptan or eletriptan produced vasocontraction as well. Hence, we concluded that the 5HT(1B)-mediated inhibition of adenylate cyclase was not a sufficient condition to couple the receptor stimulation to vasocontraction. 5. We discussed agonist-induced trafficking as a plausible mechanism for the observed phenomenon.

Animals↗

PubMed1999

Ligand efficacy and affinity in an interacting 7TM receptor model.

Quantitative understanding of the activation of G protein-coupled receptors is based mostly on some theoretical models that describe the interaction between ligand and protein partners and the activation process of the receptor. All of these models provide different definitions for observable affinity or efficacy. However, the property common to such parameters defined in the context of these models is that they are always independent of the concentration of the receptor molecule. This is based on the assumption that receptors do not interact with each other appreciably. In this article, experimental evidence for which this assumption does not seem to apply is discussed and an oligomerization model for seven-transmembrane-domain receptors that explains the relationship between receptor concentration, apparent affinity and efficacy is provided.

Humans↗