The structural determinants of G protein coupling versus activation by G protein-coupled receptors are not well understood. We examine the role of two distinct basic regions in the carboxyl terminal portion of the third intracellular loop of the alpha(2A)-adrenergic receptor to dissect these aspects of function. Changing three arginines to alanines by mutagenesis and stable expression in Chinese hamster ovary-K1 cells impaired the alpha(2)-adrenergic receptor G(s)-mediated stimulation of cyclic AMP (cAMP) accumulation, whereas G(i)-mediated inhibition was normal. When two (B2) or three (B3) basic residues closer to transmembrane span 6 were mutated to alanine, normal ligand binding was observed, but G(i)-mediated inhibition of cAMP accumulation showed 20-fold and 50-fold decreases in agonist potency for the B2 and B3 mutants, respectively. Surprisingly, a normal G(s) response was seen for the B2 mutant, and the B3 mutant showed only a 6-fold decrease in agonist potency. Mutation of both the three alanines and B3 residues to alanines showed a 200-fold decrease in agonist potency for G(i)-mediated inhibition of cAMP accumulation, whereas the G(s) response was nearly completely eliminated. The three basic residues (which include the BB of the BBXXF motif) play a role as G(i) activators rather than in receptor-G protein coupling, because high-affinity agonist binding is intact. Thus, we have identified three basic residues required for activation of G(i) but not required for receptor-G protein coupling. Also, distinct basic residues are required for optimal G(i) and G(s) responses, defining a microspecificity determinant within the carboxyl terminal portion of the third intracellular loop of the alpha(2a) adrenergic receptor.
BackgroundProblem-based learning (PBL) has become the most significant innovation in medical education of the past 40 years. In contrast to exam-centered, lecture-based conventional curricula, PBL is a comprehensive curricular strategy that fosters student-centred learning and the skills desired in physicians. The rapid spread of PBL has produced many variants. One of the most common is 'hybrid PBL' where conventional teaching methods are implemented alongside PBL. This paper contends that the mixing of these two opposing educational philosophies can undermine PBL and nullify its positive benefits. Schools using hybrid PBL and lacking medical education expertise may end up with a dysfunctional curriculum worse off than the traditional approach.DiscussionFor hybrid PBL schools with a dysfunctional curriculum, standard PBL is a cost-feasible option that confers the benefits of the PBL approach. This paper describes the signs of a dysfunctional PBL curriculum to aid hybrid PBL schools in recognising curricular breakdown. Next it discusses alternative curricular strategies and costs associated with PBL. It then details the four critical factors for successful conversion to standard PBL: dealing with staff resistance, understanding the role of lectures, adequate time for preparation and support from the administrative leadership.SummaryHybrid PBL curricula without oversight by staff with medical education expertise can degenerate into dysfunctional curricula inferior even to the traditional approach from which PBL emerged. Such schools should inspect their curriculum periodically for signs of dysfunction to enable timely corrective action. A decision to convert fully to standard PBL is cost feasible but will require time, expertise and commitment which is only sustainable with supportive leadership.
G protein-coupled receptors form a ternary complex of ligand, receptor, and G protein heterotrimer (LRG) during signal transduction from the outside to the inside of a cell. Our goal was to develop a homogeneous, small-volume, bead-based approach compatible with high-throughput flow cytometry that would allow evaluation of G protein coupled receptor molecular assemblies. Dextran beads were derivatized to carry chelated nickel to bind hexahistidine-tagged green fluorescent protein (GFP) and hexahistidine-tagged G proteins. Ternary complexes were assembled on these beads using fluorescent ligand with wild-type receptor or a receptor-Gi␣2 fusion protein, and with a nonfluorescent ligand and receptor-GFP fusion protein. Streptavidin-coated polystyrene beads used biotinylated anti-FLAG antibodies to bind FLAG-tagged G proteins for ternary complex assembly. Validation was achieved by showing time and concentration dependence of ternary complex formation. Affinity measurements of ligand for receptor on particles, of the ligand-receptor complex for G protein on the particles, and receptor-Gi␣2 fusion protein for G␥, were consistent with comparable assemblies in detergent suspension. Performance was assessed in applications representing the potential of these assemblies for ternary complex mechanisms. We showed the relationship for a family of ligands between LR and LRG affinity and characterized the affinity of both the wild-type and GFP fusion receptors with G protein. We also showed the potential of kinetic measurements to allow observation of individual steps of GTP-induced ternary complex disassembly and discriminated a fast step caused by RG disassembly compared with the slower step of G␣␥ disassembly.GPCRs interact with extracellular stimuli, such as photons, hormones, neurotransmitters, and odorants (Gilman, 1995). These stimuli cause conformational changes in the receptor, leading to binding of intracellular G protein heterotrimers, each with one copy of a guanyl nucleotide binding ␣ subunit and a ␥ dimer (Neer, 1995). After stimulation, the ␣ subunit binds GTP, which promotes dissociation of the ␣ subunit from the ␥ dimer, exposing new surfaces to cytoplasmic effectors, such as adenylyl cyclase and phospholipase C. The human genome contains ϳ600 GPCR genes, 27 ␣, 5 , and 13 ␥ (Venter et al., 2001), with smaller numbers of these G proteins (17, 5, and 12, respectively) found to date. With such large numbers, determining how productively any given GPCR couples to a particular ␣␥ heterotrimer is daunting (1020 ␣␥ combinations alone). The assembly of a high agonist-affinity complex is a good criterion of productive partners (Gilman, 1987).The formyl peptide receptor (FPR) responds to the presence of N-formyl methionine-containing peptides resulting from bacterial and mitochondrial protein synthesis, as well as other hydrophobic peptides (Gao et al., 1994). This receptor has served as a model for signal transduction in phagocytic cells and for inflammatory and autoimmune diseases (Prossnitz and Ye, 1...
G protein coupled receptors activate signal transducing guanine nucleotide-binding proteins (G proteins), which consist of an alpha subunit and a betagamma dimer. Whole cell studies have reported that receptors signal through specific betagamma subtypes. Membrane reconstitution studies with the adenosine A(1) and alpha(2A) adrenergic receptors have reached a similar conclusion. We aimed to test the generality of this finding by comparing the gamma subtype specificity for four G(i)-coupled receptors: alpha(2A) adrenergic; A1 adenosine (A(1)-R); 5-hydroxytryptamine(1A) (5-HT(1A)-R); mu opioid. Membranes were reconstituted with Galpha(i)(1) and five gamma subtypes (dimerized to beta1). Using a sensitive alpha-betagamma binding assay, we show that all recombinant betagamma (except beta1gamma1) had comparable affinity for alpha(i)(1). Using high affinity agonist binding as a measure of receptor-G protein coupling, betagamma-containing gamma11 was the most potent for A(1)-R and 5-HT(1A)-R (p < 0.05, one way ANOVA) while gamma7 was most potent for the other two receptors. gamma11 was 3-8-fold more potent for the A(1)-R than were the other gamma subtypes. Also, gamma11 was 2-8-fold more potent for A(1)-R than at the other receptors, suggesting a unique coupling specificity of the A(1)-R for gamma11. In contrast, the discrimination by receptors for the other betagamma subtypes (beta1 and gamma1, gamma2, gamma7, and gamma10) was limited (2-3-fold). Thus the exquisite betagamma specificity of individual receptors reported in whole cell studies may depend on in vivo mechanisms beyond direct receptor recognition of betagamma subtypes.
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