26 research outputs found
Structural evidence for counter-current flow in proximal tubules versus pertitubular capillaries in the rat kidney. Evaluation of the counter-current mechanism between the proximal convoluted tubules and the peritubular capillaries in the rat nephron.
Mutation of a conserved aspartic acid in the Beta2 adrenergic recptor: constitutive activation, structural instability, and conformational rearrangement of transmembrane segment VI
A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein
G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo- and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the β(2)-adrenergic receptor (β(2)AR), can be incorporated into a reconstituted high-density lipoprotein (rHDL) phospholipid bilayer particle together with the stimulatory heterotrimeric G protein, Gs. Single-molecule fluorescence imaging and FRET analysis demonstrate that a single β(2)AR is incorporated per rHDL particle. The monomeric β(2)AR efficiently activates Gs and displays GTP-sensitive allosteric ligand-binding properties. These data suggest that a monomeric receptor in a lipid bilayer is the minimal functional unit necessary for signaling, and that the cooperativity of agonist binding is due to G protein association with a receptor monomer and not receptor oligomerization
Polydipsia in the Dog—Symposium: I The Role of the Kidneys in the Control of Water Metabolism in Mammals
Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies
The biologic activity induced by ligand binding to orthosteric or allosteric sites on a G protein–coupled receptor (GPCR) is mediated by stabilization of specific receptor conformations. In the case of the β(2) adrenergic receptor, these ligands are generally small-molecule agonists or antagonists. However, a monomeric single-domain antibody (nanobody) from the Camelid family was recently found to allosterically bind and stabilize an active conformation of the β(2)-adrenergic receptor (β(2)AR). Here, we set out to study the functional interaction of 18 related nanobodies with the β(2)AR to investigate their roles as novel tools for studying GPCR biology. Our studies revealed several sequence-related nanobody families with preferences for active (agonist-occupied) or inactive (antagonist-occupied) receptors. Flow cytometry analysis indicates that all nanobodies bind to epitopes displayed on the intracellular receptor surface; therefore, we transiently expressed them intracellularly as “intrabodies” to test their effects on β(2)AR-dependent signaling. Conformational specificity was preserved after intrabody conversion as demonstrated by the ability for the intracellularly expressed nanobodies to selectively bind agonist- or antagonist-occupied receptors. When expressed as intrabodies, they inhibited G protein activation (cyclic AMP accumulation), G protein–coupled receptor kinase (GRK)–mediated receptor phosphorylation, β-arrestin recruitment, and receptor internalization to varying extents. These functional effects were likely due to either steric blockade of downstream effector (G(s), β-arrestin, GRK) interactions or stabilization of specific receptor conformations which do not support effector coupling. Together, these findings strongly implicate nanobody-derived intrabodies as novel tools to study GPCR biology
Early segmental changes in ischemic acute tubular necrosis of the rat kidney. Relationship between structural changes of the proximal convoluted tubule and its counter-current capillary vascularization
Conformational biosensors reveal GPCR signalling from endosomes
A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited or non-subcellular resolution(1). It has been subsequently proposed that signalling by internalized GPCR is restricted to G-protein-independent mechanisms such as scaffolding by arrestins(2,3), or GPCR activation elicits a discrete form of persistent G protein activation(4–9), or that internalized GPCR can indeed contribute to the acute G protein-mediated response(10). Evidence supporting these various latter hypotheses is indirect or subject to alternate interpretation, and it remains unknown if endosome-localized GPCR are even present in an active form. Here we describe the application of conformation-specific single domain antibodies (nanobodies) to directly probe activation of the β(2)-adrenoceptor, a prototypical GPCR(11), and its cognate G protein, G(s) (ref. 12) in living mammalian cells. We show that the adrenergic agonist isoprenaline promotes receptor and G protein activation in the plasma membrane as expected, but also in the early endosome membrane; and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane, and suggest a versatile strategy for probing dynamic conformational change in vivo