A key role for transmembrane prolines in calcitonin receptor-like receptor agonist binding and signalling:implications for family B G-protein-coupled receptors

Calcitonin receptor like-receptor is a family B G-protein coupled receptor (GPCR). It requires receptor activity modifying protein (RAMP) 1 to give a calcitonin gene-related peptide (CGRP) receptor. Little is known of how members of this receptor family function. Proline residues often form important kinks in alpha-helices. Therefore, all proline residues within the transmembrane helices of the receptor (Pro241, Pro244 in helix 4, Pro275 in helix 5, Pro321 and Pro331 in helix 6) were mutated to alanine. Pro241 Pro275, and Pro321 are highly conserved throughout all family B GPCRs. The binding of CGRP and its ability to stimulate cAMP production were investigated in mutant and wild-type receptors after transient transfection into COS-7 cells with RAMP1. The P321A mutation significantly decreased the pEC(50) for CGRP and reduced its affinity but did not change cell-surface expression. Antagonist binding [CGRP(8-37) and 1-piperidinecarboxamide N-[2-[[5amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3 5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quina zolinyl) (BIBN4096BS)] was little altered by the mutation. Adrenomedullin-mediated signaling was disrupted when P321A was coexpressed with RAMP1, RAMP2, or RAMP3. The P331A mutant produced a moderate reduction in CGRP binding and receptor activation. Mutation of the other residues had no effect on receptor function. Thus, Pro321 and Pro331 are required for agonist binding and receptor activation. Modeling suggested that Pro321 induces a bend in helix 6, bringing its C terminus near that of helix 3, as seen in many family A GPCRs. This is abolished in P321A. P321A-I325P predicted to restore this conformation, showed wild-type activation. Modeling can also rationalize the effects of transmembrane proline mutants previously reported for another family B GPCR, the VPAC(1) receptor.

Evidence that interaction between conserved residues in transmembrane helices 2, 3, and 7 are crucial for human VPAC1 receptor activation

The VPAC(1) receptor belongs to family B of G protein-coupled receptors (GPCR-B) and is activated upon binding of the vasoactive intestinal peptide (VIP). Despite the recent determination of the structure of the N terminus of several members of this receptor family, little is known about the structure of the transmembrane (TM) region and about the molecular mechanisms leading to activation. In the present study, we designed a new structural model of the TM domain and combined it with experimental mutagenesis experiments to investigate the interaction network that governs ligand binding and receptor activation. Our results suggest that this network involves the cluster of residues Arg(188) in TM2, Gln(380) in TM7, and Asn(229) in TM3. This cluster is expected to be altered upon VIP binding, because Arg(188) has been shown previously to interact with Asp(3) of VIP. Several point mutations at positions 188, 229, and 380 were experimentally characterized and were shown to severely affect VIP binding and/or VIP-mediated cAMP production. Double mutants built from reciprocal residue exchanges exhibit strong cooperative or anticooperative effects, thereby indicating the spatial proximity of residues Arg(188), Gln(380), and Asn(229). Because these residues are highly conserved in the GPCR-B family, they can moreover be expected to have a general role in mediating function.

A multigene family encoding R-SNAREs in the ciliate Paramecium tetraurelia

SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane interactions and are conventionally divided into Q-SNAREs and R-SNAREs according to the possession of a glutamine or arginine residue at the core of their SNARE domain. Here, we describe a set of R-SNAREs from the ciliate Paramecium tetraurelia consisting of seven families encoded by 12 genes that are expressed simultaneously. The complexity of the endomembrane system in Paramecium can explain this high number of genes. All P. tetraurelia synaptobrevins (PtSybs) possess a SNARE domain and show homology to the Longin family of R-SNAREs such as Ykt6, Sec22 and tetanus toxin-insensitive VAMP (TI-VAMP). We localized four exemplary PtSyb subfamilies with GFP constructs and antibodies on the light and electron microscopic level. PtSyb1-1, PtSyb1-2 and PtSyb3-1 were found in the endoplasmic reticulum, whereas PtSyb2 is localized exclusively in the contractile vacuole complex. PtSyb6 was found cytosolic but also resides in regularly arranged structures at the cell cortex (parasomal sacs), the cytoproct and oral apparatus, probably representing endocytotic compartments. With gene silencing, we showed that the R-SNARE of the contractile vacuole complex, PtSyb2, functions to maintain structural integrity as well as functionality of the osmoregulatory system but also affects cell division.

FREADMAN, AnneThe Livres-Souvenirs of Colette Genre and the Telling of Time Oxford: Legenda, August 2012200 pp., £40.00, ISBN: 978-1-906540-93-7

Full text: The title of the book gives us a major clue on the innovative approach developed by Anne Freadman in her analysis of a particular Colette corpus, the one devoted to auto-biographical writing: Les Vrilles de la vigne, Mes apprentissages, La Maison de Claudine, Sido ,L’E ́toile Vesper and Le Fanal bleu. Freadman follows the powerful lure of Rimbaldianvieilles vieilleries and its echoes with Colette’s fondness for collecting objects, people and memories. To this must be added a technical aspect, that of the study of the genre of Colette’s writing. Freadman argues that, by largely avoiding the autobiographical form, the writer achieves a new way of ‘telling time’, collecting anecdotes and detail taken from the quotidian and setting them within an all-encompassing preoccupation with time. This provides the second part of the title.The sonata form directs the sequence of the book, orchestrated into five parts,from ‘exposition’ to ‘first subject’ to‘bridge’ to ‘second subject’ to ‘recapitulation’. This has the advantage of enabling Freadman to move and progress between distinct themes—autobiography first,then alternative forms—with grace,whilst preserving within her own writing what she sees as the essence of Colette’s relationship to time in her ‘Livres-Souvenirs’, the telling of time. This‘telling of time’ is itself therefore cleverly subjected to the time constraints and freedoms of musical composition. Freadman’s ‘Exposition’ takes us through a discussion of the autobiographical genre, analysing the texts against anumber of theorists, from Lejeune to Benjamin and Ricoeur, before launching into ‘Colette and Autobiography’. It argues pertinently that Colette did not write a ‘sustained’ autobiography, even inthe most autobiographical of her writings, Mes apprentissages. Measured against Goodwin’s three sources for autobiography, confession, apologia and memoirs, Colette’s autobiographical writings appear to be at odds with all of them. Freadman then goes on in Part II of her argument, to persuasively uncover a project that rejects self-scrutiny and with no autobiographical strategy. In ‘Collecting Time’, despite claims of continuity, narrative logic and causality areabandoned in favour of a collection offragments, family stories that are built up generation after generation into familylegends. A close and fruitful analysis of Sidoleads us to a study of ‘The Art of Ending’, concentrating on L’E ́toile Vesperandle Fanal Bleu. The closing chapter gives a fascinating reading of La Naissance du jouras an exemplar of the way in which the two subjects developed in Freadman’s volume are cast together:Colette’s own working through the autobiographical genre, and her refusal to write memoirs, in favour of collecting memories, and the strategies she uses for her purpose. In ‘Recapitulation’, her concluding chapter, Freadman adroitlyen capsulates her analysis in a fetching title: ‘Fables of Time’. Indeed, the wholepremise of her book is to move away from autobiographical genre, having acknowledged the links and debt the corpus owes to it, and into a study of the multiple and fruitful ways in which Colette tells time.The rich and varied readings of thematerial, competently informed by theoretical input, together with acute sensitivity to the corpus, mark out this study as incontournable for Colette scholars.