Activation of heterotrimeric G proteins by their cognate seven transmembrane domain receptors is believed to involve conformational changes propagated from the receptor to the G proteins. However, the nature of these changes remains unknown. We monitored the conformational rearrangements at the interfaces between receptors and G proteins and between G protein subunits by measuring bioluminescence resonance energy transfer between probes inserted at multiple sites in receptor-G protein complexes. Using the data obtained for the alpha(2A)AR-G alpha(i1) beta1gamma2 complex and the available crystal structures of G alpha(i1) beta1gamma2, we propose a model wherein agonist binding induces conformational reorganization of a preexisting receptor-G protein complex, leading the G alpha-G betagamma interface to open but not dissociate. This conformational change may represent the movement required to allow nucleotide exit from the G alpha subunit, thus reflecting the initial activation event.

Functional selectivity of G protein-coupled receptor (GPCR) ligands toward different downstream signals has recently emerged as a general hallmark of this receptor class. However, pleiotropic and crosstalk signaling of GPCRs makes functional selectivity difficult to decode. To look from the initial active receptor point of view, we developed new, highly sensitive and direct bioluminescence resonance energy transfer-based G protein activation probes specific for all G protein isoforms, and we used them to evaluate the G protein-coupling activity of [(1)Sar(4)Ile(8)Ile]-angiotensin II (SII), previously described as an angiotensin II type 1 (AT(1)) receptor-biased agonist that is G protein independent but β-arrestin selective. By multiplexing assays sensing sequential signaling events, from receptor conformations to downstream signaling, we decoded SII as an agonist stabilizing a G protein-dependent AT(1A) receptor signaling module different from that of the physiological agonist angiotensin II, both in recombinant and primary cells. Thus, a biased agonist does not necessarily select effects from the physiological agonist but may instead stabilize and create a new distinct active pharmacological receptor entity.

This study analyzed the regulation of alpha2-adrenoceptors (alpha2-ARs) in human vascular smooth muscle cells (VSMs). Saphenous veins and dermal arterioles or VSMs cultured from them expressed high levels of alpha2-ARs (alpha2C > alpha2A, via RNase protection assay) and responded to alpha2-AR stimulation [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK-14,304, 1 microM)] with constriction or calcium mobilization. In contrast, VSMs cultured from aorta did not express alpha2-ARs and neither cultured cells nor intact aorta responded to UK-14,304. Although alpha2-ARs (alpha2C >> alpha2A) were detected in aortas, alpha2C-ARs were localized by immunohistochemistry to VSMs of adventitial arterioles and not aortic media. In contrast with aortas, aortic arterioles constricted in response to alpha2-AR stimulation. Reporter constructs demonstrated higher activities for alpha2A- and alpha2C-AR gene promoters in arteriolar compared with aortic VSMs. In arteriolar VSMs, serum increased expression of alpha2C-AR mRNA and protein but decreased expression of alpha2A-ARs. Serum induction of alpha2C-ARs was reduced by inhibition of p38 mitogen-activated protein kinase (MAPK) with 2 microM SB-202190 or dominant-negative p38 MAPK. UK-14,304 (1 microM) caused calcium mobilization in control and serum-stimulated cells: in control VSMs, the response was inhibited by the alpha2A-AR antagonist BRL-44408 (100 nM) but not by the alpha2C-AR antagonist MK-912 (1 nM), whereas after serum stimulation, MK-912 (1 nM) but not BRL-44408 (100 nM) inhibited the response. These results demonstrate site-specific expression of alpha2-ARs in human VSMs that reflects differential activity of alpha2-AR gene promoters; namely, high expression and function in venous and arteriolar VSMs but no detectable expression or function in aortic VSMs. We found that alpha2C-ARs can be dramatically and selectively induced via a p38 MAPK-dependent pathway. Therefore, altered expression of alpha2C-ARs may contribute to pathological changes in vascular function.

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A series of derivatives structurally related to biphenyline (3) was designed with the aim to modulate selectivity toward the alpha(2)-AR subtypes. The results obtained demonstrated that the presence of a correctly oriented function with positive electronic effect (+sigma) in portion X of the ligands is an important factor for significant alpha(2C)-subtype selectivity (imidazolines 5, 13, 16, and 19). Homology modeling and docking studies support experimental data and highlight the crucial role for the hydrogen bond between the pyridine nitrogen in position 3 of 5 and the NH-indole ring of Trp6.48, which is favorably oriented in the alpha(2C)-subtype, only.

Protein kinase C (PKC) is an ubiquitous enzyme that mediates intracellular signal transduction in eukaryotes. Jurkat and U937 cells were exposed to microgravity during a Space Shuttle flight and stimulated with a radiolabeled phorbol ester (3H-PDBu) that specifically activates and labels several PKC isoforms. Both the total amount of 3H-PDBu labeling per cell and the relative distribution of labeling between subcellular compartments were altered in microgravity compared to onboard and ground 1 g control samples. The amount of total phorbol ester labeling per cell was increased approximately twofold in microgravity samples when compared with onboard 1 g samples for both cell lines. The subcellular distribution of PKC in the cytosol and nuclear fractions appeared to be correlated with the applied acceleration. In both cell types the relative amount of phorbol ester labeling in the nuclear fraction decreased with applied acceleration, whereas the labeling in cytosolic fraction increased with g level. No significant differences were observed between labeling levels in the membrane fraction in both cell types. Interleukin-1beta synthesis by U937 cells was markedly decreased in microgravity when compared to the onboard 1 g control, suggesting that the observed alterations in PKC distribution may have functional consequences. The results may have important implications for the effect of gravity on cellular signal transduction.

The regulation of insulin release from pancreatic beta cells is a highly complex process, integrating glycaemia value as well as the modulatory actions of many hormones, neuropeptides and neurotransmitters [1]. The endocrine pancreas is richly innervated by sympathetic neurons and electrical stimulation of splanchnic nerves or treatment with epinephrine results in strong inhibition of insulin secretion [2]. Pharmacological studies carried out both in vivo [3±4] and on isolated islets [5] show that this inhibitory effect is primarily due to stimulation of peripheral adrenoceptors of the a 2 -type. Consistent with this, binding studies with selective radioligands showed the presence of a 2 -adrenoceptors on membranes from pancreatic islets isolated from various species [6] as well as on membranes from a rodent insulinoma [7]. The a 2 -adrenoceptor family consists of three receptor subtypes (a 2A , a 2B and a 2C ) encoded by distinct genes that do not have introns [8]. Transcripts encoding a 2A -and a 2B -subtypes were detected recently in human [9] and rat [10] pancreatic islets. On the basis of functional studies, the a 2A -subtype is, however, responsible for inhibition of insulin secretion [11±12]. Diabetologia (2000) AbstractAims/hypothesis. To study the role of the human a 2A -adrenoceptor in the regulation of insulin secretion and the maintenance of glucose homeostasis in transgenic mice overexpressing this receptor in pancreatic beta cells. Methods. A human insulin promoter/human a 2 C10-adrenoceptor chimeric gene was microinjected into mouse embryos and transgenic mice were obtained. Results. Analysis by RT-PCR showed that the expression of the transgene was restricted to pancreatic islets. Study of the binding of the a 2 -antagonist [ 3 H]RX821 002 to membrane preparations showed that islets from transgenic mice had ninefold higher a 2 -adrenoceptor density than those from controls. Immunohistological analysis showed, however, no change in the number or size of islets between control and transgenic mice. Transgenic animals had normal glycaemia and insulinaemia in basal conditions but greater hyperglycaemic and hypoinsulinaemic responses after injection of the a 2 -agonist, UK14 304. The lower blood insulin concentration detected in transgenic mice was a reflection of a stronger inhibitory effect of the a 2 -agonist on glucose-stimulated insulin secretion in transgenic islets than in controls. Furthermore, transgenic mice did not have lower glycaemia to basal values after an intraperitoneal glucose tolerance test. This defect was abolished by treatment with the a 2 -adrenoceptor antagonist, RX821 002. Conclusion/interpretation. These results provide evidence in vivo that overexpression of a 2 -adrenoceptors in beta cells can lead to impaired insulin secretion and glucose intolerance. [Diabetologia (2000) 43: 899±906] Keywords Diabetes, insulin, catecholamines, a 2 -agonist, adrenoceptor, islets, transgenic mice. Corresponding author: Dr. F. Bosch, Department of Biochemistry and Molecular Biology, Scho...

The imidazoline nucleus linked in position 2 via an oxyethylene bridge to a phenyl ring carrying an ortho substituent of moderate steric bulk provided alpha(2)-adrenergic (AR) ligands endowed with significant alpha(2C)-agonism/alpha(2A)-antagonism. Similar behavior was displayed by cirazoline (12). For their positive morphine analgesia modulation (due to alpha(2C)-AR stimulation) and sedation overcoming (due to alpha(2A)-AR antagonism), 8 and 11 might be useful as adjuvant agents in the management of pain with morphine.