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.

Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function

The first and third extracellular loops (ECL) of G protein-coupled receptors (GPCRs) have been implicated in ligand binding and receptor function. This study describes the results of an alanine/leucine scan of ECLs 1 and 3 and loop-associated transmembrane (TM) domains of the secretin-like GPCR calcitonin receptor-like receptor which associates with receptor activity modifying protein 1 to form the CGRP receptor. Leu195Ala, Val198Ala and Ala199Leu at the top of TM2 all reduced aCGRP-mediated cAMP production and internalization; Leu195Ala and Ala199Leu also reduced aCGRP binding. These residues form a hydrophobic cluster within an area defined as the "minor groove" of rhodopsin-like GPCRs. Within ECL1, Ala203Leu and Ala206Leu influenced the ability of aCGRP to stimulate adenylate cyclase. In TM3, His219Ala, Leu220Ala and Leu222Ala have influences on aCGRP binding and cAMP production; they are likely to indirectly influence the binding site for aCGRP as well as having an involvement in signal transduction. On the exofacial surfaces of TMs 6 and 7, a number of residues were identified that reduced cell surface receptor expression, most noticeably Leu351Ala and Glu357Ala in TM6. The residues may contribute to the RAMP1 binding interface. Ile360Ala impaired aCGRP-mediated cAMP production. Ile360 is predicted to be located close to ECL2 and may facilitate receptor activation. Identification of several crucial functional loci gives further insight into the activation mechanism of this complex receptor system and may aid rational drug design.

Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies

Modelling class B G-protein-coupled receptors (GPCRs) using class A GPCR structural templates is difficult due to lack of homology. The plant GPCR, GCR1, has homology to both class A and class B GPCRs. We have used this to generate a class A-class B alignment, and by incorporating maximum lagged correlation of entropy and hydrophobicity into a consensus score, we have been able to align receptor transmembrane regions. We have applied this analysis to generate active and inactive homology models of the class B calcitonin gene-related peptide (CGRP) receptor, and have supported it with site-directed mutagenesis data using 122 CGRP receptor residues and 144 published mutagenesis results on other class B GPCRs. The variation of sequence variability with structure, the analysis of polarity violations, the alignment of group-conserved residues and the mutagenesis results at 27 key positions were particularly informative in distinguishing between the proposed and plausible alternative alignments. Furthermore, we have been able to associate the key molecular features of the class B GPCR signalling machinery with their class A counterparts for the first time. These include the [K/R]KLH motif in intracellular loop 1, [I/L]xxxL and KxxK at the intracellular end of TM5 and TM6, the NPXXY/VAVLY motif on TM7 and small group-conserved residues in TM1, TM2, TM3 and TM7. The equivalent of the class A DRY motif is proposed to involve Arg(2.39), His(2.43) and Glu(3.46), which makes a polar lock with T(6.37). These alignments and models provide useful tools for understanding class B GPCR function.

2 μm mode-locked thulium doped fiber laser using tilted fiber grating

A nonlinear polarization rotation based all-fiber passively mode-locked Tm3+-doped fiber laser is demonstrated by using a 45° tilted fiber grating (TFG) as an in-line polarizer. Stable soliton pulses centered at 1992.7 nm with 2.02 nm FWHM bandwidth were produced at a repetition rate of 1.902 MHz with pulse duration of 2.2 ps and pulse energy of 74.6 pJ. With the increased pump power, the laser also can operate at noise-like regime with 18.1 nm FWHM bandwidth and pulse energy of up to 250.1 nJ. Using the same 45° TFG, both stable soliton and noise-like mode-locking centered at ∼1970 nm and ∼2050 nm, were also achieved by shortening and extending the length of Tm3+-doped fiber, respectively, exhibiting advantages of broadband and low insertion loss at 2 μm band.

Thulium-doped all-fiber mode-locked laser based on NPR and 45°-tilted fiber grating

A nonlinear polarization rotation based all-fiber passively modelocked Tm3+-doped fiber laser is demonstrated by using a 45° tilted fiber grating (TFG) as an in-line polarizer. The 45° TFG centered at 2000 nm with polarization dependent loss (PDL) of >12 dB at 1850 nm∼2150 nm range was UV inscribed for the first time in SM28 fiber using a 244 nm Ar+ continuous wave laser and a phase mask with 25 mm long uniform pitch and titled period pattern of 33.7° with respect to the fiber axis. Stable soliton pulses centered at 1992.7 nm with 2.02 nm FWHM bandwidth were produced at a repetition rate of 1.902 MHz with pulse duration of 2.2 ps and pulse energy of 74.6 pJ. As increased pump power, the laser also can operate at noise-like regime with 18.1 nm FWHM bandwidth and pulse energy of up to 250.1 nJ. Using the same 45° TFG, both stable soliton and noise-like mode-locking centered at ∼1970 nm and ∼2050 nm, were also achieved by shortening and extending the length of Tm3+-doped fiber, respectively, exhibiting advantages of broadband and low insertion loss at 2 μm band.

Wide wavelength selectable all-fiber thulium doped fiber laser between 1925 nm and 2200 nm

We demonstrate an all-fiber Tm3+-doped silica fiber laser operating at a wide selectable wavelength range by using different fiber Bragg gratings (FBGs) as wavelength selection elements. With a specifically designed high reflective (HR) FBG and the fiber end as an output coupler, the lasing in the range from 1975 nm to 2150 nm with slope efficiency of >30% can be achieved. By employing a low reflective (LR) FBG as the output coupler, the obtainable wavelengths were extended to the range between 1925 nm and 2200 nm which is the reported longest wavelength from the Tm3+-doped silica fiber lasers. Furthermore, by employing a FBG array in the laser cavity and inducing bend loss between adjacent FBGs in the array, six switchable lasing wavelengths were achieved. © 2014 Optical Society of America.