Salmeterol Efficacy and Bias in the Activation and Kinase-Mediated Desensitization of ß2-Adrenergic Receptors.

Salmeterol is a long-acting ß2-adrenergic receptor (ß2AR) agonist that is widely used as a bronchodilator for the treatment of persistent asthma and chronic obstructive pulmonary disease in conjunction with steroids. Previous studies demonstrated that salmeterol showed weak efficacy for activation of adenylyl cyclase; however, its efficacy in the complex desensitization of the ß2AR remains poorly understood. In this work, we provide insights into the roles played by the G protein-coupled receptor kinase/arrestin and protein kinase A in salmeterol-mediated desensitization through bioluminescence resonance energy transfer (BRET) studies of liganded-ß2AR binding to arrestin and through kinetic studies of cAMP turnover. First, BRET demonstrated a much reduced efficacy for salmeterol recruitment of arrestin to ß2AR relative to isoproterenol. The ratio of BRETISO/BRETSALM after 5-minute stimulation was 20 and decreased to 5 after 35 minutes, reflecting a progressive decline in BRETISO and a stable BRETSALM. Second, to assess salmeterol efficacy for functional desensitization, we examined the kinetics of salmeterol-induced cAMP accumulation (0-30 minutes) in human airway smooth muscle cells in the presence and absence of phosphodiesterase inhibition. Analysis of shaping of cAMP turnover for both agonists demonstrated significant salmeterol desensitization, although it was reduced relative to isoproterenol. Using an isoproterenol rescue protocol after either short-term (10 minutes) or long-term (2 and 14 hours) salmeterol pretreatments, we found that salmeterol progressively depressed isoproterenol stimulation but did not prevent subsequent rescue by isoproterenol and additional isoproterenol-mediated desensitization. Our findings reveal a complex efficacy for functional desensitization, demonstrating that although salmeterol shows weak efficacy for adenylyl cyclase activation and G protein-coupled receptor kinase/arrestin-mediated desensitization, it acts as a strong agonist in highly amplified protein kinase A-mediated events.

Klebsiella pneumoniae antimicrobial drug resistance, United States, 1998-2010.

We studied antimicrobial-resistant Klebsiella pneumoniae for 1998-2010 by using data from The Surveillance Network. Susceptibility results (n = 3,132,354) demonstrated significant increases in resistance to all antimicrobial drugs studied, except tetracycline. Cross-resistance among carbapenem-resistant K. pneumoniae was lower for tetracycline and amikacin.

Escherichia coli antimicrobial resistance increased faster among geriatric outpatients compared with adult outpatients in the USA, 2000-10.

OBJECTIVES: Few studies have examined Escherichia coli antimicrobial resistance across age groups over time. The objective of this study was to compare urinary E. coli antimicrobial resistance trends among adult and geriatric outpatients from 2000 to 2010. METHODS: Antimicrobial susceptibility results for E. coli urine isolates from adult (aged 16-64 years) and geriatric (aged ≥65 years) outpatients were analysed using data from The Surveillance Network Database-USA. RESULTS: Susceptibility test results from adult (n = 6 412 025) and geriatric (n = 3 395 297) outpatients showed that E. coli antimicrobial resistance increased faster among geriatric outpatients for all agents studied. The greatest increases in resistance over the study time period were for ciprofloxacin (9.4% and 23.5% increases among adult and geriatric individuals, respectively), trimethoprim/sulfamethoxazole (4.3% and 10.5%) and ampicillin (2.0% and 13.6%). CONCLUSIONS: Urinary E. coli antimicrobial resistance increased faster among geriatric outpatients than adult outpatients in the USA. Rising antimicrobial resistance disproportionately affects geriatric populations and presents a threat to public health.

Short Signature rpoB Gene Sequence to Differentiate Species in Mycobacterium abscessus Group.

Mycobacterium abscessus group (MAG) are rapidly growing acid-fast bacteria that consist of three closely related species: M. abscessus (Ma), M. bolletii (Mb), and M. massiliense (Mm). Differentiation of these species can be difficult but is increasingly requested owing to recent infectious outbreaks and their differential drug resistance. We developed a novel and rapid pyrosequencing method using short signature sequences (35 to 45 bp) at a hypervariable site in the rpoB gene to differentiate the three MAG species, along with M. chelonae (Mc), and M. immunogenum (Mi). This method was evaluated using 111 M. chelonae-abscessus complex (MCAC) isolates, including six reference strains. All isolates were successfully differentiated to the species level (69 Ma, four Mb, six Mm, 23 Mc, and nine Mi). The species identifications by this method had 100% agreement with Sanger sequencing as well as an in-silico rpoB typing method. This short signature sequencing (SSS) method is rapid (6 to 7 h), accurately differentiates MAG species, and is useful for informing antimicrobial therapy decision. IMPORTANCE Mycobacterium abscessus group (MAG) are rapidly growing acid-fast bacteria that include three species: M. abscessus, M. massiliense, and M. bolletii. These species are among the leading causes of nontuberculosis mycobacteria infections in humans but difficult to differentiate using commonly used methods. The differences of drug resistance among the species shape the treatment regimens and make it significant for them to be differentiated accurately and quickly. We developed and evaluated a novel short signature sequencing (SSS) method utilizing a gene called rpoB to differentiate the three MAG species, as well as other two species (M. chelonae and M. immunogenum). The identification results had 100% agreement with both the reference method of Sanger sequencing and rpoB typing method via a computer-simulated analysis. This SSS method was accurate and quick (6 to 7 h) for species differentiation, which will benefit patient care. The technology used for this method is affordable and easy to operate.

Quantitative modeling of GRK-mediated beta2AR regulation.

We developed a unified model of the GRK-mediated beta2 adrenergic receptor (beta2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the beta2AR; (3) beta2AR internalization; (4) recycling of the beta2AR post isoproterenol treatment; (5) beta2AR desensitization; and (6) beta2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation "seen" by the beta2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the beta2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the beta2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the beta2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.

When informed consent goes poorly: a descriptive study of medical negligence claims and patient complaints.

OBJECTIVE: To describe the frequency, characteristics, and outcomes of medicolegal disputes over informed consent. DESIGN AND SETTING: Retrospective review and analysis of negligence claims against doctors insured by Avant Mutual Group Limited and complaints lodged with the Office of the Health Services Commissioner of Victoria that alleged failures in the informed consent process and were adjudicated between 1 January 2002 and 31 December 2008. MAIN OUTCOME MEASURES: Case frequency (by medical specialty), type of allegation, type of treatment. RESULTS: A total of 481 cases alleged deficiencies in the informed consent process (218 of 1898 conciliated complaints [11.5%]; 263 of 7846 negligence claims [3.4%]). 57% of these cases were against surgeons. Plastic surgeons experienced dispute rates that were more than twice those of any other specialty or subspecialty group. 92% of cases (442/481) involved surgical procedures and 16% (77/481) involved cosmetic procedures. The primary allegation in 71% of cases was that the clinician failed to mention or properly explain risks of complications. Five treatment types - procedures on reproductive organs (12% of cases), procedures on facial features excluding eyes (12%), prescription medications (8%), eye surgery (7%) and breast surgery (7%) - accounted for 46% of all cases. CONCLUSIONS: The typical dispute over informed consent involves an operation, often cosmetic, and allegations that a particular complication was not properly disclosed. With Australian courts now looking to patient preferences in setting legal standards of care for risk disclosure, medicolegal disputes provide valuable insights for targeting both quality improvement efforts and risk management activities.

Retrospective Performance Analyses of over Two Million U.S. QuantiFERON Blood Sample Results.

Both the QuantiFERON-TB Gold Plus (QFT-Plus) and the QuantiFERON-TB Gold In-Tube (QFT-GIT) tests are interferon gamma (IFN-γ) release assays (IGRAs) intended to detect in vitro cell-mediated immune responses to Mycobacterium tuberculosis antigens. In this study, we retrospectively analyzed performance data for both the QFT-GIT and QFT-Plus test systems from over 2 million samples. QFT-Plus and QFT-GIT testing was performed as specified in the respective package inserts at 23 Quest Diagnostics sites. Blood specimens were collected from individuals in all 50 states from November 2018 through December 2019. Retrospective analyses compared the proportion of positive, indeterminate, and conversion/reversion results. The overall proportion of QFT-positive results was 7% for both the QFT-Plus and QFT-GIT. The proportion of positive results was highest for QFT-GIT (7.5%) followed by the heparin 1-tube QFT-Plus (7.2%); a lower proportion of positives was observed with the 4-tube (all four QFT tubes were used in blood collection) QFT-Plus (6.0%). The proportions of indeterminate results for the 1-tube (heparin-only tube collection) and 4-tube QFT-Plus methods were less than 1% and 4%, respectively. This study indicates a higher proportion of positive results for M. tuberculosis than data from other studies. Additionally, the proportion of indeterminate QFT results were markedly lower when the sample was transported in one lithium-heparin tube instead of direct inoculation into 4 QFT-Plus tubes at the site of blood collection. IMPORTANCE In this study, we retrospectively analyzed results from both the QFT-GIT and QFT-Plus test systems from over 2 million blood specimens. The variables analyzed were (i) QFT positivity rates among various U.S. populations, (ii) indeterminate rates among various types of blood draws and how often an indeterminate result was resolved within 30 days after the initial draw, and (iii) the association of TB1 and TB2 antigen tubes with IGRA reversion and conversion events from serial QFT testing. This is, to our knowledge, the largest QFT study representing patients from an extensive geographic coverage across the United States and U.S. territories.

Recovery and Its Dynamics of Filamentous Fungi from Clinical Specimen Cultures: An Extensive Study.

The culture method remains vital in diagnosing fungal infections, but extensive data-based evaluation of the method, especially for filamentous fungi (molds), is minimal. The purpose of this study was to characterize mold recoveries from fungal cultures and the impact of media and incubation duration. Clinical specimens for fungal cultures were submitted primarily from the eastern and central United States, and mold isolation data were prospectively collected and analyzed. A total of 1,821 molds in 59 genera were isolated from 1,687 positive specimens, accounting for approximately 5.6% of our cohort of 30,000 fungal cultures. Within 2 weeks, nearly 90% of molds and 97.3% of Aspergillus fumigatus complex were recovered (>95% confidence interval [CI]). All Mucorales fungi were recovered within 11 days of incubation. The recovery peak time was day 3 for Mucorales fungi, day 4 for hyaline molds, day 5 for dematiaceous molds, and day 7 for Onygenales fungi. The recovery of Histoplasma capsulatum and Trichophyton species in the fourth week of incubation reveals that a 3-week incubation time is insufficient. Inhibitory mold agar was the best medium for recovering all mold types among all tested specimen types, yielding nearly 78% of mold growth overall, indicating the necessity of selective medium for fungal cultures. IMPORTANCE Fungal culture is the gold standard method of diagnosing fungal infections, but important information, such as the impact of media and incubation times on fungal recovery, is not well documented. This study addressed these gaps using extensive data-based evaluation focused on molds. We identified the best medium types and incubation times for better fungal culture practice. We analyzed 1,821 molds from 1,687 positive specimens in our cohort of approximately 30,000 fungal cultures. Mold recovery peaked between 3 and 7 days of incubation, dependent upon the type of mold. Some well-defined fungal pathogens, such as Histoplasma capsulatum and Trichophyton species, were isolated in the fourth week of incubation. Inhibitory mold agar was identified as the best medium for recovering all mold types among all tested specimen sources. As we are aware, this is the largest study of fungal culture methods and supports 4 weeks of incubation for optimal mold recovery.

Role for the regulator of G-protein signaling homology domain of G protein-coupled receptor kinases 5 and 6 in beta 2-adrenergic receptor and rhodopsin phosphorylation.

Phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) is a major mechanism of desensitization of these receptors. GPCR activation of GRKs involves an allosteric site on GRKs distinct from the catalytic site. Although recent studies have suggested an important role of the N- and C-termini and domains surrounding the kinase active site in allosteric activation, the nature of that site and the relative roles of the RH domain in particular remain unknown. Based on evolutionary trace analysis of both the RH and kinase domains of the GRK family, we identified an important cluster encompassing helices 3, 9, and 10 in the RH domain in addition to sites in the kinase domain. To define its function, a panel of GRK5 and -6 mutants was generated and screened by intact-cell assay of constitutive GRK phosphorylation of the beta(2)-adrenergic receptor (beta 2AR), in vitro GRK phosphorylation of light-activated rhodopsin, and basal catalytic activity measured by tubulin phosphorylation and autophosphorylation. A number of double mutations within helices 3, 9, and 10 reduced phosphorylation of the beta2AR and rhodopsin by 50 to 90% relative to wild-type GRK, as well as autophosphorylation and tubulin phosphorylation. Based on these results, helix 9 peptide mimetics were designed, and several were found to inhibit rhodopsin phosphorylation by GRK5 with an IC(50) of approximately 30 microM. In summary, our studies have uncovered previously unrecognized functionally important sites in the regulator of G-protein signaling homology domain of GRK5 and -6 and identified a peptide inhibitor with potential for specific blockade of GRK-mediated phosphorylation of receptors.

Parkinson's Disease Caregiver Strain in Singapore.

Background: Caregiver strain is recognized globally with Parkinson's disease (PD). Comparatively little is understood about caregiver burden and strain in Asia. Objective: To investigate caregiver strain for families living with PD in Singapore, in light of international data. Methods: Ninety-four caregivers were recruited via people living with idiopathic PD in Singapore. Caregiver strain was assessed using the Zarit Burden Interview (ZBI); health status was assessing using the Cumulative Illness Rating Scale for Geriatrics (CIRS-G). PD disability measures were the Unified Parkinson's Disease Rating Scale (UPDRS) and modified Hoehn and Yahr (1967) Scale. Results: Primary caregivers of people living with PD in Singapore were mostly cohabiting spouses, partners or offspring. Around half employed foreign domestic helpers. Mean caregiving duration was 5.9 years with an average of eight hours per day spent in caregiving roles. Most care providers were comparatively healthy. Caregivers reported significant levels of strain which increased with greater level of disability (r = 0.36, n = 94, p < 0.001). Associations were significant between caregiver strain and scores on the UPDRS mentation, behavior, and mood subscales [r = 0.46, n = 94, p < 0.001, 95% CI (0.28, 0.60)]. High scores on the UPDRS activities of daily living subscale were associated with caregiver strain [r = 0.50, n = 94, p < 0.001, CI (0.33, 0.64)]. Conclusion: Most caregivers in this Singapore sample reported high levels of strain, despite comparatively good physical function. Caregiver strain in PD spans geopolitical and cultural boundaries and correlates with disease severity. These results support the need for better early recognition, education, and support for caregivers of people living with PD.

Current significance of the Mycobacterium chelonae-abscessus group.

Organisms of the Mycobacterium chelonae-abscessus group can be significant pathogens in humans. They produce a number of diseases including acute, invasive and chronic infections, which may be difficult to diagnose correctly. Identification among members of this group is complicated by differentiating at least eleven (11) known species and subspecies and complexity of identification methodologies. Treatment of their infections may be problematic due to their correct species identification, antibiotic resistance, their differential susceptibility to the limited number of drugs available, and scarcity of susceptibility testing.

Accelerated dephosphorylation of the beta2-adrenergic receptor by mutation of the C-terminal lysines: effects on ubiquitination, intracellular trafficking, and degradation.

Agonist-mediated ubiquitination regulates some G protein-coupled receptors by targeting them to lysosomes for degradation. Phosphorylation also regulates receptor endocytosis and trafficking to lysosomes. To explore the roles of the two post-translational modifications, we mutated the three C-terminal lysines to arginines in the human beta 2-adrenergic receptor (beta 2AR) (K348/372/375R). The level of agonist-mediated ubiquitination of the mutant (3K/R) was greatly reduced compared to that of wild-type (WT) beta 2AR in whole cells and in cell-free assays. Downregulation of 3K/R also was attenuated compared to that of the WT, whereas internalization and recycling were more similar. During endocytosis, WT and 3K/R appeared in different vesicles and WT, but not 3K/R, was transported to lysosomes. Both were rapidly phosphorylated in agonist-stimulated cells, but upon agonist removal, the rate of dephosphorylation of 3K/R initially was approximately 5 times faster than that of WT. The increased rate also was observed in a cell-free, soluble assay and, thus, was not due to differences in receptor trafficking. Okadaic acid, a potent phosphatase inhibitor, reduced the level of dephosphorylation and increased the levels of lysosomal targeting and degradation of 3K/R. The reduced level of ubiquitination and rapid dephosphorylation of 3K/R appear to prevent it from being sorted to lysosomes in contrast to the phosphorylated and ubiquitinated WT beta 2AR. Our findings indicate that both phosphorylation and ubiquitination are involved in the intracellular sorting of beta 2AR between pathways of recycling to the plasma membrane and degradation in lysosomes, and that the rate of dephosphorylation may be another mechanism of regulating the sorting.

Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins.

The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (a-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.

Acid-fast bacterium detection and identification from paraffin-embedded tissues using a PCR-pyrosequencing method.

AIMS: Acid-fast bacterium (AFB) identification from formalin-fixed paraffin-embedded (FFPE) tissues is challenging and may not be readily available to the clinical laboratory. A method to detect and identify AFB from FFPE tissues using PCR and pyrosequencing (PCR-Seq) was developed and evaluated. METHODS: The method was validated using spiked cell-clotted paraffin blocks before use with patients' specimens. DNA was extracted from tissue sections, and a 16S rRNA gene fragment was amplified and a signature sequence was produced on a PyroMark ID system. Sequences were aligned to established databases for AFB identification. Additional tissue sections were stained and examined for AFB. RESULTS: Both sensitivity and specificity were 100% on spiked cell-clotted blocks without cross-reactivity with non-AFB. Of 302 FFPE tissues from patients, 116 (38%) were AFB-stain positive; 83 (72%) of these had AFB identified. The 21 AFB identified included Mycobacterium tuberculosis complex (14 cases), Mycobacterium leprae (3), Mycobacterium genavense (2), Mycobacterium marinum-ulcerans group (3) and 17 other AFB (61). Thirteen cases were AFB-stain indeterminate and 4 were positive by the PCR-Seq method. Of the AFB stain-negative cases, 167 were negative and 6 were positive by PCR-Seq. CONCLUSIONS: The PCR-Seq method provided specific identification of various AFB species or complexes from FFPE tissues.

Differential phosphorylation and dephosphorylation of beta2-adrenoceptor sites Ser262 and Ser355,356.

Activated beta2-adrenoceptors are rapidly desensitized by phosphorylation of Ser262 by protein kinase A (PKA) and of Ser355,356 by G-protein-coupled receptor kinase (GRK). We sought to determine whether the phosphorylation and subsequent dephosphorylation of these sites had similar kinetics and requirements for receptor endocytosis. The phosphorylation of the PKA and GRK sites were measured using antibodies that recognize phosphoserine 262 and phosphoserine 355,356. Endocytosis in stably transfected HEK293 cells was blocked by inducible expression of dominant-negative dynamin-1 K44A or by treatment with hypertonic sucrose. The phosphorylation of the GRK site Ser355,356 during a 10 microM isoprenaline treatment rapidly reached a steady state, and the extent of kinetics of phosphorylation were unaffected by dynamin-1 K44A expression, and minimally by hypertonic sucrose. In contrast, phosphorylation of the PKA site Ser262 during a 10 microM isoprenaline treatment peaked after 2 min and then rapidly declined, while inhibition of endocytosis enhanced and prolonged phosphorylation. Treatment with 300 pM isoprenaline, a concentration too low to provoke endocytosis, also resulted in prolonged PKA site phosphorylation. The dephosphorylation of these sites was measured after removal of agonist. Significant dephosphorylation of phosphoserines 262 and 355,356 was observed under conditions of very low endocytosis, however dephosphorylation of the GRK site was greater if antagonist was present after removal of agonist. The results indicate that the kinetics of beta2-adrenoceptor GRK and PKA site phosphorylation are distinct and differently affected by endocytosis, and that receptor dephosphorylation can occur either at the plasma membrane or in internal compartments.

Beta 2 adrenergic receptor 5' haplotypes influence promoter activity.

1. Transcriptional control of the human beta(2) adrenergic receptor gene (ADRB2) predominantly resides within a 549 base pair region immediately 5' to the start of translation. Within this region, four naturally occurring polymorphisms, -468 C-->G, -367 T-->C, -47 T-->C, and -20 T-->C, have been identified. 2. To determine the individual site and haplotype effects of these polymorphisms, we generated 16 luciferase-based mutant constructs which were transiently transfected into HEK293 cells, and measured ADRB2 promoter-driven luciferase activity. 3. Two of the 16 mutant constructs, GCCT (-468G, -367C, -47C, -20T) and CTCT, showed a highly significant 3 fold decrease in luciferase induction relative to the reference CTTT. These haplotype effects could not be accounted for by the separate and additive effects of each site. 4. These findings indicate that promoter polymorphisms interact to significantly alter beta(2) adrenergic receptor expression, and should be examined further for their association with disease-related phenotypes.

Endosome sorting of beta 2-adrenoceptors is GRK5 independent.

1. We have investigated the role of G protein-coupled receptor kinase 5 (GRK5) in the regulation of endosome sorting of human beta(2)-adrenoceptors. 2. Expressing GRK5 at a high level significantly increased the extent of internalization of wild-type beta(2)-adrenoceptors and of an internalization-defective mutant receptor, and increased receptor phosphorylation at serines 355 and 356 in the cytoplasmic tail. 3. Overexpressing GRK5 did not alter beta(2)-adrenoceptor recycling as assessed by immunofluorescence microscopy and radioligand binding assays nor was there any change in receptor downregulation. 4. These data indicate that GRK5 does not regulate the sorting of beta(2)-adrenoceptors in the endocytic pathway.