Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs.

G-protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR-G protein complexes, few are in a lipid membrane environment. Here, we report cryo-EM structures of complexes of neurotensin, neurotensin receptor 1 and Gαi1ß1γ1 in two conformational states, resolved to resolutions of 4.1 and 4.2 Å. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein-protein interactions at the GPCR-G protein interface as compared to structures obtained in detergent micelles. The findings show that the lipid membrane modulates the structure and dynamics of complex formation and provide a molecular explanation for the stronger interaction between GPCRs and G proteins in lipid bilayers. We propose an allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.

Conformational transitions of a neurotensin receptor 1-Gi1 complex.

Neurotensin receptor 1 (NTSR1) is a G-protein-coupled receptor (GPCR) that engages multiple subtypes of G protein, and is involved in the regulation of blood pressure, body temperature, weight and the response to pain. Here we present structures of human NTSR1 in complex with the agonist JMV449 and the heterotrimeric Gi1 protein, at a resolution of 3 Å. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR-Gi/o complexes (in which the nucleotide-binding pocket adopts more flexible conformations that may facilitate nucleotide exchange), and a non-canonical state in which the G protein is rotated by about 45 degrees relative to the receptor and exhibits a more rigid nucleotide-binding pocket. In the non-canonical state, NTSR1 exhibits features of both active and inactive conformations, which suggests that the structure may represent an intermediate form along the activation pathway of G proteins. This structural information, complemented by molecular dynamics simulations and functional studies, provides insights into the complex process of G-protein activation.

Receptor-mediated internalization of [3H]-neurotensin in synaptosomal preparations from rat neostriatum.

Following its binding to somatodendritic receptors, the neuropeptide neurotensin (NT) internalizes via a clathrin-mediated process. In the present study, we investigated whether NT also internalizes presynaptically using synaptosomes from rat neostriatum, a region in which NT1 receptors are virtually all presynaptic. Binding of [(3)H]-NT to striatal synaptosomes in the presence of levocabastine to block NT2 receptors is specific, saturable, and has NT1 binding properties. A significant fraction of the bound radioactivity is resistant to hypertonic acid wash indicating that it is internalized. Internalization of [(3)H]-NT, like that of [(125)I]-transferrin, is blocked by sucrose and low temperature, consistent with endocytosis occurring via a clathrin-dependent pathway. However, contrary to what was reported at the somatodendritic level, neither [(3)H]-NT nor [(125)I]-transferrin internalization in synaptosomes is sensitive to the endocytosis inhibitor phenylarsine oxide. Moreover, treatment of synaptosomes with monensin, which prevents internalized receptors from recycling to the plasma membrane, reduces [(3)H]-NT binding and internalization, suggesting that presynaptic NT1 receptors, in contrast to somatodendritic ones, are recycled back to the plasma membrane. Taken together, these results suggest that NT internalizes in nerve terminals via an endocytic pathway that is related to, but is mechanistically distinct from that responsible for NT internalization in nerve cell bodies.

Identification in the rat neurotensin receptor of amino-acid residues critical for the binding of neurotensin.

In order to identify charged amino-acid residues of the cloned rat brain neurotensin (NT) receptor (NTR) that are critical for NT binding, we performed site-directed mutagenesis on the cDNA encoding this protein, followed by transient expression into mammalian COS-7 cells and in Xenopus laevis oocytes. Point substitutions of charged residues in the N-terminal part and in the 2nd and 3rd extracellular loop of the receptor either did not affect (125)I-Tyr3-NT binding or resulted in a decrease in binding affinity by a factor of 2-3. Mutations of amino acids Asp113 in the second transmembrane domain (TM) and of Arg149 or Asp150 in TM III yielded receptors that bound NT as efficiently as the native receptor. By contrast, replacement of the Asp139 residue in the 1st extracellular loop, or of Arg143 or Arg327-Arg328 residues at the top of TM III and in TM VI, respectively, completely abolished ligand binding. Confocal and EM immunocytochemical studies of the expression of these affected receptors, tagged with the C-terminal sequence of the vesicular stomatitis virus glycoprotein (VSV-G), indicated that this loss of binding was not due to altered receptor expression or to their improper insertion into the plasma membrane. When these mutated forms of neurotensin receptor were expressed into Xenopus oocytes, Asp139-Gly- and Arg143-Gly-modified receptors remained functional in spite of a lowered response to NT whereas the Arg327-Arg328 mutant form was totally insensitive to NT at concentrations up to 10 microM. In the case of the Arg327-Arg328 mutation, the observed insensibility to NT could be the result of a drastic conformational alteration of this mutant protein. By contrast, it would appear that Asp139 and Arg143 residues located in the first extracellular loop of the receptor may be directly involved in the interaction of the receptor with neurotensin.