An inactive receptor-G protein complex maintains the dynamic range of agonist-induced signaling

Agonist binding promotes activation of G protein-coupled receptors (GPCRs) and association of active receptors with G protein heterotrimers. The resulting active-state ternary complex is the basis for conventional stimulus-response coupling. Although GPCRs can also associate with G proteins before agonist binding, the impact of such preassociated complexes on agonist-induced signaling is poorly understood. Here we show that preassociation of 5-HT₇ serotonin receptors with G_s heterotrimers is necessary for agonist-induced signaling. 5-HT₇ receptors in their inactive state associate with G_s, as these complexes are stabilized by inverse agonists and receptor mutations that favor the inactive state. Inactive-state 5-HT₇–G_s complexes dissociate in response to agonists, allowing the formation of conventional agonist–5-HT₇–G_s ternary complexes and subsequent G_s activation. Inactive-state 5-HT₇–G_s complexes are required for the full dynamic range of agonist-induced signaling, as 5-HT₇ receptors spontaneously activate G_s variants that cannot form inactive-state complexes. Therefore, agonist-induced signaling in this system involves two distinct receptor-G protein complexes, a conventional ternary complex that activates G proteins and an inverse-coupled binary complex that maintains the inactive state when agonist is not present.

G protein-coupled receptors (GPCRs) transduce a wide variety of physiological signals and are targeted by a substantial fraction of all therapeutic drugs (1). GPCRs are conformationally dynamic and transition between inactive and active states, the latter being capable of interacting with and activating heterotrimeric G proteins (2). Although some level of constitutive activity is common, the conformational equilibrium “setpoint” usually favors the inactive state of the receptor, thus keeping the system turned off and ready to respond to agonists. Agonist binding stabilizes active conformations and promotes the formation of transient active-state ternary agonist-receptor-G protein complexes (3). This positive allosteric interaction between agonist and G protein binding is the hallmark of conventional GPCR coupling. Receptor-G protein complexes that form before agonist binding have also been described (4–8) and are generally thought of as a means to promote rapid or specific signaling after agonist binding. However, the properties and functional significance of such “preassociated” complexes are largely unknown, and inactive receptor conformations are generally considered unable to interact with G proteins. Here we show that unliganded 5-HT₇ serotonin receptors form complexes with G_s heterotrimers, and that these complexes help maintain the receptor in an inactive state. Agonist binding leads to dissociation of inactive-state 5-HT₇–G_s complexes, which in turn allows increased formation of active-state 5-HT₇–G_s complexes and G protein activation. Thus, a negative allosteric interaction between agonist and G protein binding is required for the full sensitivity of these receptors to serotonin.     

Slow upsloping ST-segment depression during exercise: does it really signify a positive stress test?

Background Slow upsloping ST-segment depression during stress is thought to represent an ischemic response to exercise treadmill testing (ETT). Aim We used modern single-photon emission computed tomography (SPECT) imaging protocols to determine the incidence of ischemia in patients with slow upsloping ST depression during exercise and whether this response signifies more or less severe coronary artery disease (CAD) and risk in comparison with rapid upsloping ST depression and particularly with horizontal or downsloping ST depression. Methods We enrolled 33 patients (group 1) with rapid upsloping ST depression (>1 mm extending <0.08 seconds beyond J point), 32 patients (group 2) with slow upsloping depression (>1.5 mm extending >0.08 seconds beyond J point), and 35 patients (group 3) with horizontal or downsloping depression (>1 mm at 0.08 seconds beyond J point). Summed stress score (SSS), summed difference score (SDS), stress extent percent (SE%) and reversible extent percent (RE%) of perfusion abnormalities, lung-heart ratio (LHR), and transient ischemic dilatation (TID) were calculated. Results The mean SSS, SDS, SE%, RE%, and LHR were similar between groups 1 and 2 but significantly higher in group 3. Incidence of ischemia was similar in groups 1 and 2 (39% and 25%) but significantly higher in group 3 (77%, P < .001). Evidence of TID was seen in none of the patients in groups 1, in 3% of patients in group 2, and in 23% of patients in group 3. Conclusions Slow upsloping ST depression does not signify more severe ischemia, more extensive CAD, or more stress-induced backward left ventricular failure. Thus, it would be reasonable to consider patients with slow upsloping ST depression during exercise as having a very low likelihood of CAD, similar to patients with rapid upsloping ST depression. (Am Heart J 2002;143:482-7.)     

Impact of single immunosuppressive drug withdrawal on lymphocyte immunoreactivity

Background

Chronic rejection is a major cause of graft loss in kidney transplant recipients. Nonadherence to drug therapy is a well-recognized cause of chronic rejection leading to long-term graft dysfunction and failure for transplant recipients. Immunosuppressive medications with short half-lives that require frequent dosing, such as tacrolimus, complicate transplant regimens and may increase noncompliance. Regimens could be simplified using drugs with long half-lives requiring once-daily administration, such as sirolimus. The impact of missing doses of single agents has not been studied extensively. Erratic compliance or temporary discontinuation of immunosuppressive drugs may have significant implications for chronic rejection.

Methods

Our study evaluated the impact of single drug withdrawal of commonly used immunosuppressive agents (sirolimus and tacrolimus) on lymphocyte responses. We analyzed lymphocyte proliferation, cytokine secretion, and adenosine triphosphate generation using a crossover study design with normal healthy patients. Lymphocyte proliferation was assessed using 5-bromo-2-deoxyuridine incorporation, and T cell function was analyzed by examining adenosine triphosphate generation.

Results

Our results indicate that sirolimus exerts prolonged suppression of lymphocyte proliferation and decreased interleukin 17A that lasts up to 48 h after drug withdrawal. In comparison, tacrolimus did not have a similar effect on lymphocyte proliferation or interleukin 17A secretion.

Conclusion

Future analysis of sirolimus in diverse transplantation populations merits investigation.     

A randomized phase II trial of tacrolimus,mycophenolate mofetil and sirolimus after non-myeloablative unrelated donor transplantation

The study is a randomized phase II trial investigating graft-versus-host disease prophylaxis after non-myeloablative (90 mg/m² fludarabine and 2 Gy total body irradiation) human leukocyte antigen matched unrelated donor transplantation. Patients were randomized as follows: arm 1 – tacrolimus 180 days and mycophenolate mofetil 95 days (n=69); arm 2 – tacrolimus 150 days and mycophenolate mofetil 180 days (n=71); arm 3 – tacrolimus 150 days, mycophenolate mofetil 180 days and sirolimus 80 days (n=68). All patients had sustained engraftment. Grade II-IV acute graft-versus-host disease rates in the 3 arms were 64%, 48% and 47% at Day 150, respectively (arm 3 vs. arm 1 (hazard ratio 0.62; P=0.04). Owing to the decreased incidence of acute graft-versus-host disease, systemic steroid use was lower at Day 150 in arm 3 (32% vs. 55% in arm 1 and 49% in arm 2; overall P=0.009 by hazard ratio analysis). The Day 150 incidence of cytomegalovirus reactivation was lower in arm 3 (arm 1, 54%; arm 2, 47%; arm 3, 22%; overall P=0.002 by hazard ratio analysis). Non-relapse mortality was comparable in the three arms at two years (arm 1, 26%; arm 2, 23%; arm 3, 18%). Toxicity rates and other outcome measures were similar between the three arms. The addition of sirolimus to tacrolimus and mycophenolate mofetil is safe and associated with lower incidence of acute graft-versus-host disease and cytomegalovirus reactivation. (clinicaltrials.gov identifier: 00105001).