Previous work on the desensitization of G proteincoupled receptors has focused on the role of arrestin binding following receptor phosphorylation. We have examined the hypothesis that phosphorylation alone contributes to desensitization. In this study we demonstrate that for the G q -coupled gastrin-releasing peptide receptor (GRP-R), phosphorylation by GRK2 to a stoichiometry of ϳ1 mol PO 4 /mol GRP-R is sufficient in the absence of arrestin to reduce the rate of receptor catalyzed G protein activation by approximately 80%. Furthermore, GRP-Rs exposed in vivo to agonist are rapidly phosphorylated to a similar stoichiometry and are desensitized to a similar degree. Finally, the molecular mechanism for both in vitro GRK2-induced and in vivo agonist-induced desensitization is primarily a decrease in the maximum velocity (V max ) for the catalysis of guanine nucleotide exchange by the GRP-R rather than a change in the affinity of the receptor for the ␣ q or ␤␥ subunits. Based on these results, we suggest that, for some G protein-coupled receptors, phosphorylation has a role in desensitization that is independent of arrestin.Bombesin-like peptides elicit a variety of effects including mitogenesis, hormone secretion, and modulation of neuron firing rate (1). These effects are transduced through a family of G protein-coupled receptors (GPCRs) 1 including the gastrin-releasing peptide receptor (GRP-R) (2, 3). An agonist-activated GPCR catalyzes the exchange of guanine nucleotide on the ␣ subunit of a heterotrimeric G protein leading to the formation of G␣-GTP. The subsequent dissociation of the heterotrimeric G protein leads to stimulation of signal transduction cascades mediated by the free G␣-GTP and G␤␥ subunits (4). In the bombesin receptor family, the activated GRP-R catalyzes guanine nucleotide exchange on G␣ q (5) to activate effectors including phospholipase C-␤ (3).Mechanisms to attenuate receptor signaling have evolved that limit the amplitude and/or duration of the signal transduction cascade(s) and are collectively referred to as "desensitization." At the molecular level, desensitization may result from the degradation of ligand, from changes in receptor availability or activity, or from changes in the availability or activity of downstream effector molecules (6). Rhodopsin, which signals through transducin (G t ), and the ␤ 2 -adrenergic receptor (␤ 2 AR), which signals through G s , have been the most extensively studied GPCRs. In both cases, rapid agonist-induced receptor phosphorylation along with subsequent binding of an arrestin to the receptor play critical roles in receptor deactivation (7,8). Many other GPCRs are also known to be rapidly phosphorylated after addition of agonist (9, 10), including the GRP-R (11). Two classes of protein kinase have been implicated in agonist-induced GPCR phosphorylation: 1) the second messenger-dependent protein kinases A and C and 2) the second messenger independent kinases, called G protein-coupled receptor kinases (GRKs), including rhodopsin kinase (RK or GRK1) an...

We have used surface plasmon resonance (SPR) measurements for the kinetic analysis of G-protein-receptor interaction monitored in real time. Functionally active rhodopsin was immobilized on an SPR surface, with full retention of biochemical specific activity for catalysis of nucleotide exchange on the retinal G-protein alpha subunit, via binding to immobilized concanavalin A. The binding interactions of bovine retinal alpha(t) and beta(1)gamma(1) subunits with rhodopsin measured by SPR were profoundly synergistic. Synergistic binding of the retinal G-protein subunits to rhodopsin was not observed for guanosine 5'-[gamma-thio]triphosphate-bound Galpha(t), nor was binding observed with squid retinal Galpha(q), which is not activated by bovine rhodopsin. The binding affinity (336+/-171 nM; mean value+/-S.D.) of retinal betagamma for rhodopsin in the presence of retinal alpha subunit measured by SPR confirmed the apparent affinity of 254 nM determined previously by nucleotide exchange assays. Binding of beta(1)gamma(1), beta(1)gamma(2), and beta(1)gamma(8-olf) dimers to rhodopsin, independently of the alpha subunit, was readily observable by SPR. Further, these dimers, differing only in their gamma subunit compositions, displayed markedly distinct binding affinities and kinetics. The beta(1)gamma(2) dimer bound with a kinetically determined K(d) of 13+/-3 nM, a value nearly identical with the biochemically determined K(1/2) of 10 nM. The physiologically appropriate beta(1)gamma(1) displayed rapid association and dissociation kinetics, whereas the other beta(1)gamma dimers dissociated at a rate less than 1/100 as fast. Thus rhodopsin interaction with its native signalling partners is both rapid and transient, whereas the interaction of rhodopsin with heterologous Gbetagamma dimers is markedly prolonged. These results suggest that the duration of a G-protein-coupled receptor signalling event is an intrinsic property of the G-protein coupling partners; in particular, the betagamma dimer.

We have used surface plasmon resonance (SPR) measurements for the kinetic analysis of G-protein-receptor interaction monitored in real time. Functionally active rhodopsin was immobilized on an SPR surface, with full retention of biochemical specific activity for catalysis of nucleotide exchange on the retinal G-protein α subunit, via binding to immobilized concanavalin A. The binding interactions of bovine retinal αt and β1γ1 subunits with rhodopsin measured by SPR were profoundly synergistic. Synergistic binding of the retinal G-protein subunits to rhodopsin was not observed for guanosine 5′-[γ-thio]triphosphate-bound Gαt, nor was binding observed with squid retinal Gαq, which is not activated by bovine rhodopsin. The binding affinity (336±171nM; mean value±S.D.) of retinal βγ for rhodopsin in the presence of retinal α subunit measured by SPR confirmed the apparent affinity of 254nM determined previously by nucleotide exchange assays. Binding of β1γ1, β1γ2, and β1γ8-olf dimers to rhodopsin, independently of the α subunit, was readily observable by SPR. Further, these dimers, differing only in their γ subunit compositions, displayed markedly distinct binding affinities and kinetics. The β1γ2 dimer bound with a kinetically determined Kd of 13±3nM, a value nearly identical with the biochemically determined K1/2 of 10nM. The physiologically appropriate β1γ1 displayed rapid association and dissociation kinetics, whereas the other β1γ dimers dissociated at a rate less than 1/100 as fast. Thus rhodopsin interaction with its native signalling partners is both rapid and transient, whereas the interaction of rhodopsin with heterologous Gβγ dimers is markedly prolonged. These results suggest that the duration of a G-protein-coupled receptor signalling event is an intrinsic property of the G-protein coupling partners; in particular, the βγ dimer.

Drug development is a resource and time-intensive process resulting in attrition rates of up to 90%. As a result, repurposing existing drugs with established safety and pharmacokinetic profiles is gaining traction as a way of accelerating therapeutics development. Here we have developed unique machine learning-driven Natural Language Processing and biomedical semantic technologies that mine over 53 million biomedical documents to automate the generation of a 911M edge knowledge graph. We then applied subgraph queries that relate drugs to diseases using genetic evidence to identify potential drug repurposing candidates for a broad range of diseases. We use Carney Complex, a disease with no known treatment, to illustrate our approach. This analysis revealed Ruxolitinib (Incyte, trade name Jakafi), a JAK1/2 inhibitor with an established safety and efficacy profile approved to treat myelofibrosis, as a potential candidate for the treatment of Carney Complex through off-target drug activity.

A 57 year-old Caucasian male was initially admitted to Methodist Hospital from an outside hospital for dehydration, increased heart rate, and weight loss. He has a past medical history of coronary artery disease status post myocardial infarction and coronary artery bypass graft, congestive heart failure, paroxysmal atrial fibrillation on coumadin, diabetes type II on insulin, and an orthotopic liver transplant in 1997 for end-stage liver disease secondary to hepatitis C virus and alpha-1 antitrypsin disorder.At the outside hospital, his one-month hospital course was complicated by acute on chronic renal failure which warranted hemodialysis, anemia that required transfusions, MRSA line sepsis from his Perm-A-Cath treated with vancomycin, and pneumonia, which was possibly secondary to aspiration. The patient was transferred to our facility for further management.Additional past medical history is significant for hyperlipidemia, depression and C3 to C5 fracture. Additional surgeries include AICD placement, a C3 to C4 diskectomy, cholecystectomy and appendectomy. Social history was negative for alcohol and substance abuse, but positive for a 30 pack year smoking history, which he quit one year ago. Family history was significant for diabetes, coronary artery disease and lung cancer. Current medications included amiodarone, aspirin, carvedilol, nexium, zetia, insulin, nebulizers, sirolimus, vancomycin, zosyn, and gentamicin. His only allergy was to meperidine.Initial vital signs were temperature 98.9˚ Fahrenheit, pulse 77 beats/minutes, respirations 16 breaths/minute, blood pressure 126/70 mm Hg, oxygen saturation 99 % on room air. The patient was in no acute distress. Physical exam was significant only for bilateral crackles at the lung bases, multiple skin tattoos, a rightsided Perm-A-Cath in the chest that was non-tender without surrounding erythema or swelling, and a surgical scar in the right upper quadrant.Chest radiograph on admission showed a right lower lobe consolidation, and he was continued on antibiotics for presumed pneumonia. The patient appeared to be improving after finishing his course of antibiotics for line sepsis and suspected pneumonia, and plans were made to transfer him to a subacute rehab with outpatient dialysis. One week after hospital transfer, he developed fevers and chills with accompanying shortness of breath. Blood cultures grew out pan-sensitive E. coli from his Perm-A-Cath. The catheter was removed and a Shiley catheter was placed. After completing a course of antibiotics, he remained afebrile, and when his blood cultures showed no growth for 48 hours, another Perm-A-Cath was placed. However, he became febrile again. A repeat chest radiograph showed increasing left lingular pneumonia in addition to an unchanged right lower lobe consolidation (Figure 1). At this point, he finished a course of empiric moxifloxacin which was recommended by the infectious disease service.He continued to have intermittent fevers, shortness of breath, a non-productive cough, and hypoxia that required oxyge...