Antisera of designed specificity for subunits of guanine nucleotide-binding regulatory proteins.

Mumby, Susanne M.; Kahn, Richard; Manning, David R.; Gilman, Alfred G.

doi:10.1073/pnas.83.2.265

Cited by 347 publications

(167 citation statements)

References 25 publications

Supporting

Mentioning

160

Contrasting

Order By: Relevance

“…It was also shown in preliminary experiments that the antibody does not label non-specific bands in non-infected Sf9 cells. In order to assess G protein binding to the purified receptor, the blots were incubated with nonselective anti-G protein α-subunit antisera (A 569, 1:1000 dilution, a generous gift form Dr. Susan Mumby (Mumby et al, 1986)). Immunoreactive bands were labeled with horseradish peroxidase-conjugated secondary antibodies and visualized using the SuperSignal WestDura Extended Duration Substrate system (Pierce, Rockford, IL).…”

Section: Sds/page and Western Analysismentioning

confidence: 99%

Unique agonist-bound cannabinoid CB1 receptor conformations indicate agonist specificity in signaling

Georgieva

Devanathan

Stropova

et al. 2008

European Journal of Pharmacology

View full text Add to dashboard Cite

Cannabinoid drugs differ in their rank order of potency to produce analgesia versus other central nervous system effects. We propose that these differences are due to unique agonist-bound cannabinoid CB 1 receptor conformations that exhibit different affinities for individual subsets of intracellular signal transduction pathways. In order to test this hypothesis, we have used plasmonwaveguide resonance (PWR) spectroscopy, a sensitive method that can provide direct information about ligand-protein and protein-protein interactions, and can detect conformational changes in lipidembedded proteins. A recombinant epitope-tagged human cannabinoid CB 1 receptor was expressed in insect Sf9 cells, solubilized and purified using two-step affinity chromatography. The purified receptor was incorporated into a lipid bilayer on the surface of the PWR resonator. PWR spectroscopy demonstrated that cannabinoid agonists exhibit high affinity (K D = 0.2 ± 0.03 nM and 2 ± 0.4 nM for CP 55,940 and WIN 55,212-2, respectively) for the purified epitope tagged hCB 1 receptor. Interestingly however, these structurally different cannabinoid agonists shifted the PWR spectra in opposite directions, indicating that CP 55,940 and WIN 55,212-2 binding leads to different hCB 1 receptor conformations. Furthermore, PWR experiments also indicated that these CP 55,940-and WIN 55,212 -bound hCB 1 receptor conformations exhibit slightly different affinities to an inhibitory G protein heterotrimer, G i1 (K D = 27 ± 8 nM and K D = 10.7 ± 4.7 nM, respectively), whereas they strikingly differ in their ability to activate this G protein type.

show abstract

Section: Sds/page and Western Analysismentioning

confidence: 99%

Unique agonist-bound cannabinoid CB1 receptor conformations indicate agonist specificity in signaling

Georgieva

Devanathan

Stropova

et al. 2008

European Journal of Pharmacology

View full text Add to dashboard Cite

show abstract

“…Thus, even though the primary amino acid sequences of the retinal and brain are the same by peptide mapping [41,49], immunological reactivity towards a peptidedirected antibody [38] and cloning [50,51], their amino termini are blocked (Codina, J., Cook, R. and Birnbaumer, L., unpublished results). This indicates the existence of at least one posttranslational modification, the chemical nature of which is unknown and which could vary with the cell type.…”

Section: Discussionmentioning

confidence: 99%

Studies on the interaction of α subunits of GTP‐binding proteins with βγ dimers

Graf

Mattera

Codina

et al. 1992

European Journal of Biochemistry

View full text Add to dashboard Cite

The interaction of several preparations of purified p;, dimers with two types of guanosinenucleotide-binding-regulatory-(G)-protein a subunits, a recombinant bvai3, made in Sf9 Spuduptern frugiperdu cells by the baculovirus (bv) expression system, and as, either purified from human erythrocyte G,-type GTP-binding protein, and activated by NaF/A1C13, or unpurified as found in a natural membrane, were studied. The By dimers used were from bovine rod outer segments (ROS), bovine brain, human erythrocytes (hRBC) and human placenta and contained distinct ratios of p subunits that, upon electrophoresis, migrated as two bands with approximate M , of 35000 and 36000, as well as distinct complements of at least two ; J subunits each. When tested for their ability to rccombinc at submaximal concentrations with bv,xi3, ROS, brain, hRBC and placental py dimers exhibited apparent affinities that were the same within a factor of two. When bovine brain, placental and ROS /?y dimers were tested for their ability to promote deactivation of G,, brain and placental by dimers were equipotent and at least 10-fold more potent than that of KOS p1~ dimers; likewise, brain by and placental dimers were equipotent in inhibiting GTP-activated and GTP-plus-isoproterenolactivated adenylyl cyclase, while ROS by dimers were less potent when assayed at the same concentration. The possibility that different CI subunits may distinguish subsets of By dimers from a single cell was investigated by analyzing the by composition of three G proteins, G,, Gi2 and Gi3, purified to near homogeneity from a single cell type, the human erythrocyte. No evidence for an a-subunitspecific difference in fly composition was found. These findings suggests that, in most cells, a subunits interact indistinctly with a common pool of / +$ dimers. However, since at least one By preparation (ROS) showed unique behavior, it is clear that there may be mechanisms by which some combinations of py dimers may exhibit selectivity for the a subunits they interact with.One of the characteristics of the trimeric signal transducing heterotrimetric GTP-binding proteins of apy subunit composition (G proteins) is that they can be activated by GTP analogs such as guanosine 5'-ly-thio]triphosphate (GTP[yS]) to give two reaction products: a complex of the guanine nucleotide with the Y subunit and a free By dimer.Although not yet proven unequivocally, the dissociation reaction is believed to occur also in membranes containing GTP, undcr thc catalytic effect of ligand-activated receptors [l , 21. It has been speculated [2] and proposed [3, 41 that both a subunits and fly dimers may regulate effector functions under physiologic conditions. Such a role has indeed been firmly established for a subunits, which act at picomolar concentrations in several

show abstract

“…The transfer of proteins to nitrocellulose membranes and the immunodetection of the Sst subtypes 1-4 (sst1-4) or the ␣i 1-3 G protein subunits using specific antibodies, were carried out as described elsewhere (Mumby et al, 1986). Briefly, after protein transfer, the nitrocellulose membranes were pre-incubated with blotting buffer [50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.05% (v/v) Tween-20, and 5% (w/v) non-fat dry milk].…”

Section: Immunodetection Of Sst Subtypes and ␣I Subunits Of G Proteinsmentioning

confidence: 99%

A vitamin A-free diet results in impairment of the rat hippocampal somatostatinergic system

2006

View full text Add to dashboard Cite

Abstract-Previous studies have revealed the presence of retinoid specific receptors in the hippocampus and have demonstrated that vitamin A deficiency produces a severe deficit in spatial learning and memory which are linked to a proper hippocampal functioning. It is also well known that the tetradecapeptide somatostatin binds to specific receptors in the hippocampus and, when injected into this brain area, facilitates the acquisition of spatial tasks. In addition, depletion of somatostatin by cysteamine impairs acquisition of these tasks. Taken together, these studies support the idea that the hippocampal somatostatinergic system might be regulated by vitamin A. Hence, we evaluated the effects of vitamin A deprivation and subsequent administration of vitamin A on the rat hippocampal somatostatinergic system. Rats fed a vitamin A-free diet exhibited a significant reduction of somatostatin-like immunoreactivity content in the hippocampus whereas the somatostatin mRNA levels were unaltered. Vitamin A deficiency increased the somatostatin receptor density and its dissociation constant. Functional Gi activity as well as the capacity of somatostatin to inhibit basal and forskolin-stimulated adenylyl cyclase activity was decreased in vitamin A deficiency rats as compared with the control animals. All these parameters were fully restored when vitamin A was replaced in the diet. Furthermore, we found that the Gi␣ 1 , Gi␣ 2 and Gi␣ 3 protein levels were unaltered in hippocampal membranes from rats fed a vitamin A-free diet whereas subsequent vitamin A administration to these rats caused a significant increase in the levels of Gi␣1 and Gi␣2. Altogether, the present findings suggest that dietary vitamin A levels modulate the somatostatinergic system in the rat hippocampus.

show abstract

Antisera of designed specificity for subunits of guanine nucleotide-binding regulatory proteins.

Cited by 347 publications

References 25 publications

Unique agonist-bound cannabinoid CB1 receptor conformations indicate agonist specificity in signaling

Unique agonist-bound cannabinoid CB1 receptor conformations indicate agonist specificity in signaling

Studies on the interaction of α subunits of GTP‐binding proteins with βγ dimers

A vitamin A-free diet results in impairment of the rat hippocampal somatostatinergic system

Contact Info

Product

Resources

About