Candace J. Sabers

The immunosuppressive drug, rapamycin, interferes with an undefined signaling pathway required for the progression of G1-phase T-cells into S phase. Genetic analyses in yeast indicate that binding of rapamycin to its intracellular receptor, FKBP12, generates a toxic complex that inhibits cell growth in G1 phase. These analyses implicated two related proteins, TOR1 and TOR2, as targets of the FKBP12-rapamycin complex in yeast. In this study, we have used a glutathione S-transferase (GST)-FKBP12-rapamycin affinity matrix to isolate putative mammalian targets of rapamycin (mTOR) from tissue extracts. In the presence of rapamycin, immobilized GST-FKBP12 specifically precipitates similar high molecular mass proteins from both rat brain and murine T-lymphoma cell extracts. Binding experiments performed with rapamycin-sensitive and -resistant mutant clones derived from the YAC-1 T-lymphoma cell line demonstrate that the GST-FKBP12-rapamycin complex recovers significantly lower amounts of the candidate mTOR from rapamycin-resistant cell lines. The latter results suggest that mTOR is a relevant target of rapamycin in these cells. Finally, we report the isolation of a full-length mTOR cDNA that encodes a direct ligand for the FKBP12-rapamycin complex. The deduced amino acid sequence of mTOR displays 42 and 45% identity to those of yeast TOR1 and TOR2, respectively. These results strongly suggest that the FKBP12-rapamycin complex interacts with homologous ligands in yeast and mammalian cells and that the loss of mTOR function is directly related to the inhibitory effect of rapamycin on G1- to S-phase progression in T-lymphocytes and other sensitive cell types.

FK506, an immunosuppressant that prolongs allograft survival, is a co-drug with its intracellular receptor, FKBP12. The FKBP12.FK506 complex inhibits calcineurin, a critical signaling molecule during T-cell activation. FKBP12 was, until recently, the sole FKBP known to mediate calcineurin inhibition at clinically relevant FK506 concentrations. The best characterized cellular function of FKBP12 is the modulation of ryanodine receptor isoform-1, a component of the calcium release channel of skeletal muscle sarcoplasmic reticulum. Recently, a novel protein, FKBP12.6, was found to inhibit calcineurin at clinically relevant FK506 concentrations. We have cloned the cDNA encoding human FKBP12.6 and characterized the protein. In transfected Jurkat cells, FKBP12.6 is equivalent to FKBP12 at mediating the inhibitory effects of FK506. Upon binding rapamycin, FKBP12.6 complexes with the 288-kDa mammalian target of rapamycin. In contrast to FKBP12, FKBP12.6 is not associated with ryanodine receptor isoform-1 but with the distinct ryanodine receptor isoform-2 in cardiac muscle sarcoplasmic reticulum. Our results suggest that FKBP12.6 has both a unique physiological role in excitation-contraction coupling in cardiac muscle and the potential to contribute to the immunosuppressive and toxic effects of FK506 and rapamycin.

UNLABELLED: The goal of this study was to determine whether the preoperative diagnosis of obstructive sleep apnea (OSA) is an independent risk factor for perioperative complications in patients undergoing nonotorhinolaryngologic outpatient surgical procedures. We used existing databases to identify 234 patients with polysomnography-confirmed OSA who had outpatient surgical procedures in the years 1997 through 2000. Control patients were matched for type of anesthesia, age, sex, body mass index, surgical procedure, and surgical date. Their perioperative medical records were reviewed. There was no significant difference in the intraoperative management of OSA and control patients, except that the laryngeal mask airway was less likely to be used in OSA patients. There was no significant difference in the rate of unplanned hospital admissions (23.9% versus 18.8%; odds ratio, 1.4; 95% confidence interval, 0.8-2.5) or other adverse events (2.1% versus 1.3%; odds ratio, 1.7; 95% confidence interval, 0.4-7.0) between OSA and non-OSA patients. Further, when admission did occur, it was generally unrelated to cardiac or respiratory events. In this retrospective analysis, the preoperative diagnosis of OSA was not a risk factor for either unanticipated hospital admission or for other adverse events among patients undergoing outpatient surgical procedures in a tertiary referral center. IMPLICATIONS: In patients scheduled for outpatient surgery in a large academic practice, the diagnosis of obstructive sleep apnea confirmed by polysomnography was not an independent risk factor for unanticipated hospital admission or for other adverse perioperative events.