Biomedical subjects

Paola Molinari

Publications and source records attributed to Paola Molinari.

6 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↗

PubMed2005

Different structural requirements for the constitutive and the agonist-induced activities of the beta2-adrenergic receptor.

We converted Ser-207, located in helix 5 of the beta2-adrenergic receptor, into all other natural amino acids. To quantify receptor activation as a receptor number-independent parameter and directly related to G(s) activation, we expressed the mutants in a G alpha(s)-tethered form. GTP exchange in such constructs is restricted to the fused alpha-subunit and is a linear function of the receptor concentration. Except S207R, all other mutants were expressed to a suitable level for investigation. All mutations reduced the binding affinities of the catechol agonists, epinephrine and isoproterenol, and the extent of reduction was unrelated to the residue ability to form hydrogen bonds. Instead, both enhancements and reductions of affinity were observed for the partial agonist halostachin and the antagonist pindolol. The mutations also enhanced and diminished ligand-induced receptor activation, but the effects were strictly ligand-specific. Polar residues such as Asp and His exalted the activation by full agonists but suppressed that induced by the partial agonists halostachin and dichloroisoproterenol. In contrast, hydrophobic residues such as Ile and Val augmented partial agonist activation. Only Ile and Lys produced a significant increase of constitutive activity. The effects on binding and activity were not correlated, nor did such parameters show any clear correlation with up to 78 descriptors of amino acid physicochemical properties. Our data question the idea that Ser-207 is exposed to the polar crevice in the unbound receptor. They also suggest that the active receptor form induced by a full agonist might be substantially different from that caused by constitutive activation.

Adrenergic beta-2 Receptor Agonists↗

PubMed2004

"Induced-fit" mechanism for catecholamine binding to the beta2-adrenergic receptor.

We engineered single and multiple mutations of serines 203, 204, and 207 in the fifth transmembrane domain of the beta(2)-adrenergic receptor, a region known to interact with hydroxyl groups of the catechol ring. Using such mutants, we measured the binding affinities of a panel of six catecholamine agonists differing only in the presence of substituents in the ethanolamine tail of the molecule. Although all ligands shared an intact catechol ring, they exhibited different losses of binding energy in response to the mutations. For all mutations, we found a clear relationship between the loss of binding caused by receptor mutation and that caused by the ligand modification. This indicates that the catechol ring and the ethanolamine tail synergistically influence their respective interactions when binding to the receptor. To verify this idea by a formal thermodynamic test, we used a double-mutant cycle analysis. We compared the effects of each receptor mutation with those induced by the modifications of the ligand's tail. Because such changes disrupt interactions occurring at different receptor domains, they should produce cumulative losses. In contrast, we found positive cooperativity between such effects. This means that the binding of each side of the catecholamine can enhance the binding of the other, through an effect that is probably propagated via a conformational change. We suggest that the agonist-binding pocket is not rigid but is dynamically formed as the ligand builds an increasing number of contacts with the receptor.

Amino Acid Substitution↗

PubMed2003

Expression of OP4 (ORL1, NOP1) receptors in vascular endothelium.

Endothelial cells from rat brain microvessels, human aortic artery and human umbilical vein were examined, together with ex vivo rat brain capillaries and rat aortic ring sections, for the expression of opioid receptor-like OP-4 mRNA and protein. High levels of mRNA expression and an immunopositive reaction for the receptor protein were detected in the endothelial cells from primary and from established in vitro cultures, as well as in the intima of ex vivo rat aortic rings, where the signal was limited to the endothelial layer. Interaction of the OP4 receptor with its physiological ligand nociceptin caused, in cultured endothelial cells, the activation of a mitogen-activated protein (MAP) kinase cascade. Taken together, these results show that the OP4 receptor is synthesised and functionally expressed in endothelial cells, presumably as a starting point for some vasoactive mechanism(s).

Animals↗

PubMed2003

Promiscuous coupling at receptor-Galpha fusion proteins. The receptor of one covalent complex interacts with the alpha-subunit of another.

Fusion proteins between heptahelical receptors (GPCR) and G protein alpha-subunits show enhanced signaling efficiency in transfected cells. This is believed to be the result of molecular proximity, because the interaction between linked modules of one protein chain, if not constrained by structure, should be strongly favored compared with the same in which partners react as free species. To test this assumption we made a series of fusion proteins (type 1 and 4 opioid receptors with G(o) and beta(2) adrenergic and dopamine 1 receptors with G(sL)) and some mutated analogs carrying different tags and defective GPCR or Galpha subunits. Using cotransfection experiments with readout protocols able to distinguish activation at fused and non-fused alpha-subunits, we found that both the GPCR and the Galpha limb of one fusion protein can freely interact with non-fused proteins and the tethered partners of a neighboring fusion complex. Moreover, a bulky polyanionic inhibitor can suppress with identical potency receptor-Galpha interaction, either when occurring between latched domains of a fused system or separate elements of distinct molecules, indicating that the binding surfaces are equally accessible in both cases. These data demonstrate that there is no entropy drive from the linked condition of fusion proteins and suggest that their signaling may result from the GPCR of one complex interacting with the alpha-subunit of another. Moreover, the enhanced coupling efficiency commonly observed for fusion proteins is not due to the receptor tether, but to the transmembrane helix that anchors Galpha to the membrane.

Animals↗

PubMedDate not supplied

A technique to decrease breast shape deformity during muscle contraction in submuscular augmentation mammaplasty.

The main objective of this study was to decrease breast shape distortion during pectoralis muscle contraction following submuscular augmentation mammaplasty. We followed 348 patients who had retromuscular augmentation mammoplasty: 251 (72.1%) had polyurethane-covered gel-filled, 97 (27.9%) had textured-silicone gel-filled implants. Among the 348, 46 had surgery following the Regnault technique and 302 had surgery by the below-mentioned technique. Periareolar incision, bipartision of breast parenchyma down to the fascia, undermining of breast base from the fascia downward to the inframammary sulcus or a little below it, detaching of muscle off the thoracic cage, disinsertion of abdomino-costal pectoralis attachments. Full thickness incision of pectoralis muscle on a vertical line on the nipple projection for 2-5 inches. Placing of prosthesis. Drainage. Closure. No objective evaluation was used, only clinical judgments by three observers--the surgeon, a nurse and the patient herself. The results showed a definite decrease of the dynamic deformity among patients in whom the author's technical variation was carried out. This technique allows also, for decreasing the upward pushing of the implant during pectoralis muscle contraction and facilitates stretch of the breast tissue in patients with tighter breast envelopes. Despite lack of precise measurements, conclusions drawn from clinical judgments, taken as objectively as possible, suggest that the use of this technique may offer the solution of an otherwise disturbing collateral effect, frequently seen after this operation.

Breast Implantation↗