Casein kinase II sites in the intracellular C-terminal domain of the thyrotropin-releasing hormone receptor and chimeric gonadotropin-releasing hormone receptors contribute to beta-arrestin-dependent internalization.

We have previously shown that the mammalian gonadotropin-releasing hormone receptor (GnRHR), a unique G-protein-coupled receptor (GPCR) lacking an intracellular carboxyl tail (C-tail), does not follow a beta-arrestin-dependent internalization pathway. However, internalization of a chimeric GnRHR with the thyrotropin-releasing hormone receptor (TRHR) C-tail does utilize beta-arrestin. Here, we have investigated the sites within the intracellular C-tail domain that are important for conferring beta-arrestin-dependent internalization. In contrast to the chimeric GnRHR with a TRHR C-tail, a chimeric GnRHR with the catfish GnRHR C-tail is not beta-arrestin-dependent. Sequence comparisons between these chimeric receptors show three consensus phosphorylation sites for casein kinase II (CKII) in the TRHR C-tail but none in the catfish GnRHR C-tail. We thus investigated a role for CKII sites in determining GPCR internalization via beta-arrestin. Sequential introduction of three CKII sites into the chimera with the catfish C-tail (H354D,A366E,G371D) resulted in a change in the pattern of receptor phosphorylation and beta-arrestin-dependence, which only occurred when all three sites were introduced. Conversely, mutation of the putative CKII sites (T365A,T371A,S383A) in the C-tail of a beta-arrestin-sensitive GPCR, the TRHR, resulted in decreased receptor phosphorylation and a loss of beta-arrestin-dependence. Mutation of all three CKII sites was necessary before a loss of beta-arrestin-dependence was observed. Visualization of beta-arrestin/GFP redistribution confirmed a loss or gain of beta-arrestin sensitivity for receptor mutants. Internalization of receptors without C-tail CKII sites was promoted by a phosphorylation-independent beta-arrestin mutant (R169E), suggesting that these receptors do not contain the necessary phosphorylation sites required for beta-arrestin-dependent internalization. Apigenin, a specific CKII inhibitor, blocked the increase in receptor internalization by beta-arrestin, thus providing further support for the involvement of CKII. This study presents evidence of a novel role for C-tail CKII consensus sites in targeting these GPCRs to the beta-arrestin-dependent pathway.

Constitutive and agonist-dependent homo-oligomerization of the thyrotropin-releasing hormone receptor. Detection in living cells using bioluminescence resonance energy transfer.

The ability of G-protein-coupled receptors (GPCRs) to interact to form new functional structures, either forming oligomers with themselves or forming associations with other intracellular proteins, has important implications for the regulation of cellular events; however, little is known about how this occurs. Here, we have employed a newly emerging technology, bioluminescence resonance energy transfer (BRET), used to study protein-protein interactions in living cells, to demonstrate that the thyrotropin-releasing hormone receptor (TRHR) forms constitutive homo-oligomers. This formation of TRHR homo-oligomers in the absence of ligand was shown by demonstration of an energy transfer between TRHR molecules fused to either donor, Renilla luciferase (Rluc) or acceptor, enhanced yellow fluorescent protein (EYFP) molecules. This interaction was shown to be specific, since energy transfer was not detected between co-expressed tagged TRHRs and either complementary tagged gonadotropin-releasing hormone (GnRH) or beta(2)-adrenergic receptors. Furthermore, generation of a BRET signal between the TRHRs could only be inhibited by co-expression of the wild-type TRHR and not by other GPCRs. Agonist stimulation led to a time- and dose-dependent increase in the amount of energy transfer. Inhibition of receptor internalization by co-expression of dynamin mutant K44A did not affect the interaction between TRHRs, suggesting that clustering of receptors within clathrin-coated pits is not sufficient for energy transfer to occur. BRET also provided evidence for the agonist-induced oligomerization of another GPCR, the GnRH receptor (GnRHR), and the presence of an agonist-induced interaction of the adaptor protein, beta-arrestin, with TRHR and the absence of an interaction of beta-arrestin with GnRHR. This study supports the usefulness of BRET as a powerful tool for studying GPCR aggregations and receptor/protein interactions in general and presents evidence that the functioning unit of TRHRs exists as homomeric complexes.

Elimination of "hook-effect" in two-site immunoradiometric assays by kinetic rate analysis.

Two-site or sandwich immunoradiometric assays (IRMAs) offer theoretical advantages over competitive immunoassay systems for sensitivity, precision, and rapid incubation. The practical realization of these advantages has been limited by the phenomenon of the "high-dose hook effect," such that high concentrations of an analyte give similar responses to those of much lower concentrations. We have developed a kinetic rate monitoring IRMA system for use with the Kineti-Count 48TM, an automated kinetic radioassay analyzer, which eliminates "hook-effect" interference, thereby permitting optimal assay design for increasing sensitivity and reducing incubation time. Practical illustration of these concepts is demonstrated by a 10-min, automated, quantitative assay we developed for human choriogonadotropin. The assay can detect as little as 1.2 int. units/L and kinetically screens for the hook effect. Kinetic rate analysis of the two-site IRMA and potentially of nonisotopic counterparts permits improvements in the speed and reliability of these immunoassays.

The palatopharyngoplasty operation for snoring and sleep apnea: an interim report.

We performed palatopharyngoplasty operations on 155 patients as of June 1, 1983, and have 4-month clinical follow-ups on 123 patients. Forty-nine have had repeat polysomnograms (through September 1983) that continue to show the operation is about 50% effective in curing or considerably improving obstructive sleep apnea. Symptomatic (clinical) results in these same patients are much better than 50% and may be the reason why we have had considerable difficulty in obtaining sleep study follow-ups. Snoring was eliminated or much improved in 93% of all patients and 95% of patients without serious obstructive sleep apnea. Daytime symptoms, if any, were improved in 64%. Some form of preoperative sleep study is mandatory in all patients. Two thirds of patients who present with the complaint of snoring and none of the classic symptoms of apnea actually do have total airway obstructions during sleep.

A surgical treatment for snoring and obstructive sleep apnea.

Snoring caused by oropharyngeal obstruction and some cases of obstructive sleep apnea syndrome can be cured or considerably lessened by resecting redundant tissue from patients' oropharynx and soft palate. Preoperative, and in some instances postoperative, sleep monitoring is a necessary part of evaluating these conditions.

Use of a solid-state multihead gamma counter in a second-generation system for solid-phase immunoassay.

Simultaneous advances in detector technology and solid-phase separation systems, as well as the availability of powerful desktop computers, have made possible the development of "second-generation" solid-phase immunoassays. These retain the advantages of classical solid phase while significantly accelerating reaction kinetics. Hapten assays--such as for digoxin, thyroxin, and triiodothyronine uptake--in batches of 48 are processed in about 20 min from reagent introduction until hard-copy printout, with minimal operator involvement. The system also functions as a 48-detector gamma counter, capable of counting and reducing data for any 125I-based RIA that can be run in a 12 X 75 mm test tube. System control, data management, and computer screen displays of kinetic data are provided by an unmodified Hewlett Packard HP-87XM computer. User-friendly disc-based software facilitates the creation and storage of counting and data reduction protocols for as many as 30 RIAs from various manufacturers as well as up to 30 of our own assays.

Glial fibrillary acidic (GFA) protein in Schwann cells: fact or artifact?

Antisera to the glial fibrillary acidic (GFA) protein stained a subpopulation of Schwann cells in cryostat sections of rat sciatic nerve by indirect immunofluorescence and by the peroxidase-antiperoxidase (PAP) procedure. The staining pattern was entirely different from that obtained with vimentin antisera, which uniformly decorated endoneurial tubes. Electron microscopic examination of sciatic nerve provided a possible explanation for the relatively small number of Schwann cells decorated by GFA antisera: 10 nm filaments were mainly confined to Schwann cell processes surrounding nonmyelinated axons. A marked increase in GFA-positive Schwann cells and in Schwann cells containing filaments by electron microscopy was observed in sciatic nerves undergoing Wallerian degeneration. Conversely, immunochemical procedures failed to demonstrate the presence of antigen reacting with GFA antisera in extracts of sciatic nerve, both normal and degenerated. These include absorption experiments, double immunodiffusion, immunoaffinity chromatography, and immunoradiometric assay. Two explanations may be considered for these findings: i) Schwann cell intermediate filaments and GFA protein share common antigenic determinants, the immunohistological methods being more sensitive to detect cross-reactivity as compared to immunochemical procedures on tissue extracts; and ii) the binding of anti-GFA to Schwann cell 10 nm filaments is not due to immunological cross-reactivity.

Sleep-wake disorders in the elderly: polysomnographic analysis.

The experience with 83 patients aged 60 or older from the Stanford Sleep-Wake Disorders Clinic is compared with that in 423 younger clinic patients seen during the same two-year period. Each patient received a medical, psychologic and polysomnographic evaluation. The final diagnoses were recorded according to the Diagnostic Classification System of the Association of Sleep Disorders Centers. The most common major diagnoses in the elderly group were sleep apnea syndrome (39 percent) and periodic movements-restless legs syndrome (18 percent). These syndromes showed a significantly greater prevalence in the older than in the younger patients (p less than .001), and were found in 68 percent of the elderly group. The elderly manifested more objective signs of sleep disturbance, including more wake time after sleep onset, and more frequent and longer awakenings; moreover, fewer of them experienced stage-4 sleep. The diagnostic findings seemed to indicate that complaints about sleep-wake functioning in many elderly patients may be a result of specific pathologic sleep disturbances.

Severe kyphoscoliosis, breathing, and sleep: the "Quasimodo" syndrome during sleep.

Five adult subjects with severe kyphoscoliosis were monitored during sleep. Several types of breathing abnormalities, including obstructive apnea and hypopnea, were noted. The lowest oxygen desaturations occurred during rapid eye movement (REM) sleep. Arterial pressure, continuously measured in one subject, progressively increased throughout the night in association with abnormal breathing. The use of a cuirass ventilator did not improve the nocturnal problem.

Disorders of excessive daytime somnolence: polygraphic and clinical data for 100 patients.

A consecutive series of 100 sleep apnea free patients with the complaint of excessive daytime somnolence (EDS) were evaluated; data from medical histories, physical examination, personality inventories, and polysomnography [nocturnal polysomnography (NPSG) and daytime multiple sleep latency testing (MSLT)] were tabulated. Significant differences were found between narcoleptic and non-narcoleptic patients in a number of parameters, including EDS severity, mean sleep latency on MSLT, sleep latency on NPSG, latency to REM sleep at night, number of REM sleep at night, number of REM sleep segments throughout the night, the total number of nocturnal myoclonic jerks (as well as the number occurring per hour of NREM and REM sleep), and the number of arousals and wake periods preceded by a myoclonic jerk. Significant differences in sleep latency during MSLT and NPSG testing were found between different EDS diagnostic groups of non-narcoleptic patients. The majority of patients in the MSLT group with long sleep latencies were in the diagnostic groups of EDS associated with psychophysiological and/or psychiatric problems or with drug abuse; patients with a diagnosis of idiopathic central nervous system hypersomnia or EDS associated with disturbed nocturnal sleep formed the majority of the MSLT group with short sleep latencies. The non-narcoleptic patients in a MSLT group with short sleep latencies had significantly shorter sleep latencies at night, more sleep cycles, higher sleep efficiency, and earlier REM sleep than patients with long sleep latencies.

Blind man living in normal society has circadian rhythms of 24.9 hours.

A psychologically normal blind man, living and working in normal society, suffered from a severe cyclic sleep-wake disorder. Investigations showed that he had circadian rhythms of body temperature, alertness, performance, cortisol secretion, and urinary electrolyte excretion which were desynchronized from the 24-hour societal schedule. These rhythms all had periods which were longer than 24 hours and indistinguishable from the period of the lunar day.