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Membrane proteins are of fundamental importance to cellular processes and nano-encapsulation strategies that preserve their native lipid bilayer environment are particularly attractive for studying and exploiting these proteins. Poly(styrene-co-maleic acid) (SMA) and related polymers poly(styrene-co-(N-(3-N′,N′-dimethylaminopropyl)maleimide)) (SMI) and poly(diisobutylene-alt-maleic acid) (DIBMA) have revolutionised the study of membrane proteins by spontaneously solubilising membrane proteins direct from cell membranes within nanoscale discs of native bilayer called SMA lipid particles (SMALPs), SMILPs and DIBMALPs respectively. This systematic study shows for the first time, that conformational changes of the encapsulated protein are dictated by the solubilising polymer. The photoactivation pathway of rhodopsin (Rho), a G-protein-coupled receptor (GPCR), comprises structurally-defined intermediates with characteristic absorbance spectra that revealed conformational restrictions with styrene-containing SMA and SMI, so that photoactivation proceeded only as far as metarhodopsin-I, absorbing at 478 nm, in a SMALP or SMILP. In contrast, full attainment of metarhodopsin-II, absorbing at 382 nm, was observed in a DIBMALP. Consequently, different intermediate states of Rho could be generated readily by simply employing different SMA-like polymers. Dynamic light-scattering and analytical ultracentrifugation revealed differences in size and thermostability between SMALP, SMILP and DIBMALP. Moreover, encapsulated Rho exhibited different stability in a SMALP, SMILP or DIBMALP. Overall, we establish that SMA, SMI and DIBMA constitute a ‘toolkit’ of solubilising polymers, so that selection of the appropriate solubilising polymer provides a spectrum of useful attributes for studying membrane proteins.

Membrane proteins are of fundamental importance to cellular processes and nano-encapsulation strategies that preserve their native lipid bilayer environment are particularly attractive for studying and exploiting these proteins. Poly(styrene-co-maleic acid) (SMA) and related polymers poly(styrene-co-(N-(3-N′,N′-dimethylaminopropyl)maleimide)) (SMI) and poly(diisobutylene-alt-maleic acid) (DIBMA) have revolutionised the study of membrane proteins by spontaneously solubilising membrane proteins direct from cell membranes within nanoscale discs of native bilayer called SMA lipid particles (SMALPs), SMILPs and DIBMALPs respectively. This systematic study shows for the first time, that conformational changes of the encapsulated protein are dictated by the solubilising polymer. The photoactivation pathway of rhodopsin (Rho), a G-protein-coupled receptor (GPCR), comprises structurally-defined intermediates with characteristic absorbance spectra that revealed conformational restrictions with styrene-containing SMA and SMI, so that photoactivation proceeded only as far as metarhodopsin-I, absorbing at 478 nm, in a SMALP or SMILP. In contrast, full attainment of metarhodopsin-II, absorbing at 382 nm, was observed in a DIBMALP. Consequently, different intermediate states of Rho could be generated readily by simply employing different SMA-like polymers. Dynamic light-scattering and analytical ultracentrifugation revealed differences in size and thermostability between SMALP, SMILP and DIBMALP. Moreover, encapsulated Rho exhibited different stability in a SMALP, SMILP or DIBMALP. Overall, we establish that SMA, SMI and DIBMA constitute a ‘toolkit’ of solubilising polymers, so that selection of the appropriate solubilising polymer provides a spectrum of useful attributes for studying membrane proteins.

Membrane proteins are of fundamental importance to cellular processes and nano-encapsulation strategies that preserve their native lipid bilayer environment are particularly attractive for studying and exploiting these proteins. Poly(styrene-co-maleic acid) (SMA) and related polymers poly(styrene-co-(N-(3-N′,N′-dimethylaminopropyl)maleimide)) (SMI) and poly(diisobutylene-alt-maleic acid) (DIBMA) have revolutionised the study of membrane proteins by spontaneously solubilising membrane proteins direct from cell membranes within nanoscale discs of native bilayer called SMA lipid particles (SMALPs), SMILPs and DIBMALPs respectively. This systematic study shows for the first time, that conformational changes of the encapsulated protein are dictated by the solubilising polymer. The photoactivation pathway of rhodopsin (Rho), a G-protein-coupled receptor (GPCR), comprises structurally-defined intermediates with characteristic absorbance spectra that revealed conformational restrictions with styrene-containing SMA and SMI, so that photoactivation proceeded only as far as metarhodopsin-I, absorbing at 478 nm, in a SMALP or SMILP. In contrast, full attainment of metarhodopsin-II, absorbing at 382 nm, was observed in a DIBMALP. Consequently, different intermediate states of Rho could be generated readily by simply employing different SMA-like polymers. Dynamic light-scattering and analytical ultracentrifugation revealed differences in size and thermostability between SMALP, SMILP and DIBMALP. Moreover, encapsulated Rho exhibited different stability in a SMALP, SMILP or DIBMALP. Overall, we establish that SMA, SMI and DIBMA constitute a ‘toolkit’ of solubilising polymers, so that selection of the appropriate solubilising polymer provides a spectrum of useful attributes for studying membrane proteins.

Membrane proteins are of fundamental importance to cellular processes and nano-encapsulation strategies that preserve their native lipid bilayer environment are particularly attractive for studying and exploiting these proteins. Poly(styrene-co-maleic acid) (SMA) and related polymers poly(styrene-co-(N-(3-N′,N′-dimethylaminopropyl)maleimide)) (SMI) and poly(diisobutylene-alt-maleic acid) (DIBMA) have revolutionised the study of membrane proteins by spontaneously solubilising membrane proteins direct from cell membranes within nanoscale discs of native bilayer called SMA lipid particles (SMALPs), SMILPs and DIBMALPs respectively. This systematic study shows for the first time, that conformational changes of the encapsulated protein are dictated by the solubilising polymer. The photoactivation pathway of rhodopsin (Rho), a G-protein-coupled receptor (GPCR), comprises structurally-defined intermediates with characteristic absorbance spectra that revealed conformational restrictions with styrene-containing SMA and SMI, so that photoactivation proceeded only as far as metarhodopsin-I, absorbing at 478 nm, in a SMALP or SMILP. In contrast, full attainment of metarhodopsin-II, absorbing at 382 nm, was observed in a DIBMALP. Consequently, different intermediate states of Rho could be generated readily by simply employing different SMA-like polymers. Dynamic light-scattering and analytical ultracentrifugation revealed differences in size and thermostability between SMALP, SMILP and DIBMALP. Moreover, encapsulated Rho exhibited different stability in a SMALP, SMILP or DIBMALP. Overall, we establish that SMA, SMI and DIBMA constitute a ‘toolkit’ of solubilising polymers, so that selection of the appropriate solubilising polymer provides a spectrum of useful attributes for studying membrane proteins.

The adenosine 2A receptor (A2AR), a G-protein-coupled receptor (GPCR), was solubilised and purified encapsulated in styrene maleic acid lipid particles (SMALPs). The purified A2AR-SMALP was associated with phospholipids characteristic of the plasma membrane of Pichia pastoris, the host used for its expression, confirming that the A2AR-SMALP encapsulated native lipids. The fluorescence spectrum of the A2AR-SMALP showed a characteristic broad emission peak at 330 nm, produced by endogenous Trp residues. The inverse agonist ZM241385 caused 30% increase in fluorescence emission, unusually accompanied by a red-shift in the emission wavelength. The emission spectrum also showed sub-peaks at 321 nm, 335 nm and 350 nm, indicating that individual Trp inhabited different environments following ZM241385 addition. There was no effect of the agonist NECA on the A2AR-SMALP fluorescence spectrum. Substitution of two Trp residues by Tyr suggested that ZM241385 affected the environment and mobility of Trp2466.48 in TM6 and Trp2687.33 at the extracellular face of TM7, causing transition to a more hydrophobic environment. The fluorescent moiety IAEDANS was site-specifically introduced at the intracellular end of TM6 (residue 2316.33) to report on the dynamic cytoplasmic face of the A2AR. The inverse agonist ZM241385 caused a concentration-dependent increase in fluorescence emission as the IAEDANS moved to a more hydrophobic environment, consistent with closing the G-protein binding crevice. NECA generated only 30% of the effect of ZM241385. This study provides insight into the SMALP environment; encapsulation supported constitutive activity of the A2AR and ZM241385-induced conformational transitions but the agonist NECA generated only small effects.

The adenosine 2A receptor (A2AR), a G-protein-coupled receptor (GPCR), was solubilised and purified encapsulated in styrene maleic acid lipid particles (SMALPs). The purified A2AR-SMALP was associated with phospholipids characteristic of the plasma membrane of Pichia pastoris, the host used for its expression, confirming that the A2AR-SMALP encapsulated native lipids. The fluorescence spectrum of the A2AR-SMALP showed a characteristic broad emission peak at 330 nm, produced by endogenous Trp residues. The inverse agonist ZM241385 caused 30% increase in fluorescence emission, unusually accompanied by a red-shift in the emission wavelength. The emission spectrum also showed sub-peaks at 321 nm, 335 nm and 350 nm, indicating that individual Trp inhabited different environments following ZM241385 addition. There was no effect of the agonist NECA on the A2AR-SMALP fluorescence spectrum. Substitution of two Trp residues by Tyr suggested that ZM241385 affected the environment and mobility of Trp2466.48 in TM6 and Trp2687.33 at the extracellular face of TM7, causing transition to a more hydrophobic environment. The fluorescent moiety IAEDANS was site-specifically introduced at the intracellular end of TM6 (residue 2316.33) to report on the dynamic cytoplasmic face of the A2AR. The inverse agonist ZM241385 caused a concentration-dependent increase in fluorescence emission as the IAEDANS moved to a more hydrophobic environment, consistent with closing the G-protein binding crevice. NECA generated only 30% of the effect of ZM241385. This study provides insight into the SMALP environment; encapsulation supported constitutive activity of the A2AR and ZM241385-induced conformational transitions but the agonist NECA generated only small effects.

The fundamental importance of membrane proteins in cellular processes has driven a marked increase in the use of membrane mimetic approaches for studying and exploiting these proteins. Nano-encapsulation strategies which preserve the native lipid bilayer environment are particularly attractive. Consequently, the use of poly(styrene co-maleic acid) (SMA) has been widely adopted to solubilise proteins directly from cell membranes by spontaneously forming "SMA Lipid Particles" (SMALPs). G-protein-coupled receptors (GPCRs) are ubiquitous "chemical switches", are central to cell signalling throughout the evolutionary tree, form the largest family of membrane proteins in humans and are a major drug discovery target. GPCR-SMALPs that retain binding capability would be a versatile platform for a wide range of down-stream applications. Here, using the adenosine A2A receptor (A2AR) as an archetypical GPCR, we show for the first time the utility of fluorescence correlation spectroscopy (FCS) to characterise the binding capability of GPCRs following nano-encapsulation. Unbound fluorescent ligand CA200645 exhibited a monophasic autocorrelation curve (dwell time, τD = 68 ± 2 μs; diffusion coefficient, D = 287 ± 15 μm2 s-1). In the presence of A2AR-SMALP, bound ligand was also evident (τD = 625 ± 23 μs; D = 30 ± 4 μm2 s-1). Using a non-receptor control (ZipA-SMALP) plus competition binding confirmed that this slower component represented binding to the encapsulated A2AR. Consequently, the combination of GPCR-SMALP and FCS is an effective platform for the quantitative real-time characterisation of nano-encapsulated receptors, with single molecule sensitivity, that will have widespread utility for future exploitation of GPCR-SMALPs in general.

The fundamental importance of membrane proteins in cellular processes has driven a marked increase in the use of membrane mimetic approaches for studying and exploiting these proteins. Nano-encapsulation strategies which preserve the native lipid bilayer environment are particularly attractive. Consequently, the use of poly(styrene co-maleic acid) (SMA) has been widely adopted to solubilise proteins directly from cell membranes by spontaneously forming "SMA Lipid Particles" (SMALPs). G-protein-coupled receptors (GPCRs) are ubiquitous "chemical switches", are central to cell signalling throughout the evolutionary tree, form the largest family of membrane proteins in humans and are a major drug discovery target. GPCR-SMALPs that retain binding capability would be a versatile platform for a wide range of down-stream applications. Here, using the adenosine A2A receptor (A2AR) as an archetypical GPCR, we show for the first time the utility of fluorescence correlation spectroscopy (FCS) to characterise the binding capability of GPCRs following nano-encapsulation. Unbound fluorescent ligand CA200645 exhibited a monophasic autocorrelation curve (dwell time, τD = 68 ± 2 μs; diffusion coefficient, D = 287 ± 15 μm2 s-1). In the presence of A2AR-SMALP, bound ligand was also evident (τD = 625 ± 23 μs; D = 30 ± 4 μm2 s-1). Using a non-receptor control (ZipA-SMALP) plus competition binding confirmed that this slower component represented binding to the encapsulated A2AR. Consequently, the combination of GPCR-SMALP and FCS is an effective platform for the quantitative real-time characterisation of nano-encapsulated receptors, with single molecule sensitivity, that will have widespread utility for future exploitation of GPCR-SMALPs in general.

The adenosine 2A receptor (A2AR), a G-protein-coupled receptor (GPCR), was solubilised and purified encapsulated in styrene maleic acid lipid particles (SMALPs). The purified A2AR-SMALP was associated with phospholipids characteristic of the plasma membrane of Pichia pastoris, the host used for its expression, confirming that the A2AR-SMALP encapsulated native lipids. The fluorescence spectrum of the A2AR-SMALP showed a characteristic broad emission peak at 330 nm, produced by endogenous Trp residues. The inverse agonist ZM241385 caused 30% increase in fluorescence emission, unusually accompanied by a red-shift in the emission wavelength. The emission spectrum also showed sub-peaks at 321 nm, 335 nm and 350 nm, indicating that individual Trp inhabited different environments following ZM241385 addition. There was no effect of the agonist NECA on the A2AR-SMALP fluorescence spectrum. Substitution of two Trp residues by Tyr suggested that ZM241385 affected the environment and mobility of Trp2466.48 in TM6 and Trp2687.33 at the extracellular face of TM7, causing transition to a more hydrophobic environment. The fluorescent moiety IAEDANS was site-specifically introduced at the intracellular end of TM6 (residue 2316.33) to report on the dynamic cytoplasmic face of the A2AR. The inverse agonist ZM241385 caused a concentration-dependent increase in fluorescence emission as the IAEDANS moved to a more hydrophobic environment, consistent with closing the G-protein binding crevice. NECA generated only 30% of the effect of ZM241385. This study provides insight into the SMALP environment; encapsulation supported constitutive activity of the A2AR and ZM241385-induced conformational transitions but the agonist NECA generated only small effects.

The adenosine 2A receptor (A2AR), a G-protein-coupled receptor (GPCR), was solubilised and purified encapsulated in styrene maleic acid lipid particles (SMALPs). The purified A2AR-SMALP was associated with phospholipids characteristic of the plasma membrane of Pichia pastoris, the host used for its expression, confirming that the A2AR-SMALP encapsulated native lipids. The fluorescence spectrum of the A2AR-SMALP showed a characteristic broad emission peak at 330 nm, produced by endogenous Trp residues. The inverse agonist ZM241385 caused 30% increase in fluorescence emission, unusually accompanied by a red-shift in the emission wavelength. The emission spectrum also showed sub-peaks at 321 nm, 335 nm and 350 nm, indicating that individual Trp inhabited different environments following ZM241385 addition. There was no effect of the agonist NECA on the A2AR-SMALP fluorescence spectrum. Substitution of two Trp residues by Tyr suggested that ZM241385 affected the environment and mobility of Trp2466.48 in TM6 and Trp2687.33 at the extracellular face of TM7, causing transition to a more hydrophobic environment. The fluorescent moiety IAEDANS was site-specifically introduced at the intracellular end of TM6 (residue 2316.33) to report on the dynamic cytoplasmic face of the A2AR. The inverse agonist ZM241385 caused a concentration-dependent increase in fluorescence emission as the IAEDANS moved to a more hydrophobic environment, consistent with closing the G-protein binding crevice. NECA generated only 30% of the effect of ZM241385. This study provides insight into the SMALP environment; encapsulation supported constitutive activity of the A2AR and ZM241385-induced conformational transitions but the agonist NECA generated only small effects.

Corticotrophin releasing factor (CRF) acts via two family B G-protein-coupled receptors, CRFR1 and CRFR2. Additional subtypes exist due to alternative splicing. CRFR1α is the most widely expressed subtype and lacks a 29-residue insert in the first intracellular loop that is present in CRFR1β. It has been shown previously that co-expression of CRFR1β with receptor activity modifying protein 2 (RAMP2) in HEK 293S cells increased the cell-surface expression of both proteins suggesting a physical interaction as seen with RAMPs and calcitonin receptor-like receptor (CLR). This study investigated the ability of CRFR1α, CRFR1β and CRFR2β to promote cell-surface expression of FLAG-tagged RAMP2. Four different cell-lines were utilised to investigate the effect of varying cellular context; COS-7, HEK 293T, HEK 293S and [ΔCTR]HEK 293 (which lacks endogenous calcitonin receptor). In all cell-lines, CRFR1α and CRFR1β enhanced RAMP2 cell-surface expression. The magnitude of the effect on RAMP2 was dependent on the cell-line ([ΔCTR]HEK 293 > COS-7 > HEK 293T > HEK 293S). RT-PCR indicated this variation may relate to differences in endogenous RAMP expression between cell types. Furthermore, pre-treatment with CRF resulted in a loss of cell-surface FLAG-RAMP2 when it was co-expressed with CRFR1 subtypes. CRFR2β co-expression had no effect on RAMP2 in any cell-line. Molecular modelling suggests that the potential contact interface between the extracellular domains of RAMP2 and CRF receptor subtypes is smaller than that of RAMP2 and CRL, the canonical receptor:RAMP pairing, assuming a physical interaction. Furthermore, a specific residue difference between CRFR1 subtypes (glutamate) and CRFR2β (histidine) in this interface region may impair CRFR2β:RAMP2 interaction by electrostatic repulsion.

Corticotrophin releasing factor (CRF) acts via two family B G-protein-coupled receptors, CRFR1 and CRFR2. Additional subtypes exist due to alternative splicing. CRFR1α is the most widely expressed subtype and lacks a 29-residue insert in the first intracellular loop that is present in CRFR1β. It has been shown previously that co-expression of CRFR1β with receptor activity modifying protein 2 (RAMP2) in HEK 293S cells increased the cell-surface expression of both proteins suggesting a physical interaction as seen with RAMPs and calcitonin receptor-like receptor (CLR). This study investigated the ability of CRFR1α, CRFR1β and CRFR2β to promote cell-surface expression of FLAG-tagged RAMP2. Four different cell-lines were utilised to investigate the effect of varying cellular context; COS-7, HEK 293T, HEK 293S and [ΔCTR]HEK 293 (which lacks endogenous calcitonin receptor). In all cell-lines, CRFR1α and CRFR1β enhanced RAMP2 cell-surface expression. The magnitude of the effect on RAMP2 was dependent on the cell-line ([ΔCTR]HEK 293 > COS-7 > HEK 293T > HEK 293S). RT-PCR indicated this variation may relate to differences in endogenous RAMP expression between cell types. Furthermore, pre-treatment with CRF resulted in a loss of cell-surface FLAG-RAMP2 when it was co-expressed with CRFR1 subtypes. CRFR2β co-expression had no effect on RAMP2 in any cell-line. Molecular modelling suggests that the potential contact interface between the extracellular domains of RAMP2 and CRF receptor subtypes is smaller than that of RAMP2 and CRL, the canonical receptor:RAMP pairing, assuming a physical interaction. Furthermore, a specific residue difference between CRFR1 subtypes (glutamate) and CRFR2β (histidine) in this interface region may impair CRFR2β:RAMP2 interaction by electrostatic repulsion.

The fundamental importance of membrane proteins in drug discovery has meant that membrane mimetic systems for studying membrane proteins are of increasing interest. One such system has been the amphipathic, negatively charged poly(styrene-co-maleic acid) (SMA) polymer to form “SMA Lipid Particles” (SMALPs) which have been widely adopted to solubilize membrane proteins directly from the cell membrane. However, SMALPs are only soluble under basic conditions and precipitate in the presence of divalent cations required for many downstream applications. Here, we show that the positively charged poly(styrene-co-maleimide) (SMI) forms similar nanoparticles with comparable efficiency to SMA, whilst remaining functional at acidic pH and compatible with high concentrations of divalent cations. We have performed a detailed characterization of the performance of SMI that enables a direct comparison with similar data published for SMA. We also demonstrate that SMI is capable of extracting proteins directly from the cell membrane and can solubilize functional human G-protein coupled receptors (GPCRs) expressed in cultured HEK 293T cells. “SMILPs” thus provide an alternative membrane solubilization method that successfully overcomes some of the limitations of the SMALP method.

The fundamental importance of membrane proteins in drug discovery has meant that membrane mimetic systems for studying membrane proteins are of increasing interest. One such system has been the amphipathic, negatively charged poly(styrene-co-maleic acid) (SMA) polymer to form “SMA Lipid Particles” (SMALPs) which have been widely adopted to solubilize membrane proteins directly from the cell membrane. However, SMALPs are only soluble under basic conditions and precipitate in the presence of divalent cations required for many downstream applications. Here, we show that the positively charged poly(styrene-co-maleimide) (SMI) forms similar nanoparticles with comparable efficiency to SMA, whilst remaining functional at acidic pH and compatible with high concentrations of divalent cations. We have performed a detailed characterization of the performance of SMI that enables a direct comparison with similar data published for SMA. We also demonstrate that SMI is capable of extracting proteins directly from the cell membrane and can solubilize functional human G-protein coupled receptors (GPCRs) expressed in cultured HEK 293T cells. “SMILPs” thus provide an alternative membrane solubilization method that successfully overcomes some of the limitations of the SMALP method.

The fundamental importance of membrane proteins in drug discovery has meant that membrane mimetic systems for studying membrane proteins are of increasing interest. One such system has been the amphipathic, negatively charged poly(styrene-co-maleic acid) (SMA) polymer to form “SMA Lipid Particles” (SMALPs) which have been widely adopted to solubilize membrane proteins directly from the cell membrane. However, SMALPs are only soluble under basic conditions and precipitate in the presence of divalent cations required for many downstream applications. Here, we show that the positively charged poly(styrene-co-maleimide) (SMI) forms similar nanoparticles with comparable efficiency to SMA, whilst remaining functional at acidic pH and compatible with high concentrations of divalent cations. We have performed a detailed characterization of the performance of SMI that enables a direct comparison with similar data published for SMA. We also demonstrate that SMI is capable of extracting proteins directly from the cell membrane and can solubilize functional human G-protein coupled receptors (GPCRs) expressed in cultured HEK 293T cells. “SMILPs” thus provide an alternative membrane solubilization method that successfully overcomes some of the limitations of the SMALP method.

The fundamental importance of membrane proteins in drug discovery has meant that membrane mimetic systems for studying membrane proteins are of increasing interest. One such system has been the amphipathic, negatively charged poly(styrene-co-maleic acid) (SMA) polymer to form “SMA Lipid Particles” (SMALPs) which have been widely adopted to solubilize membrane proteins directly from the cell membrane. However, SMALPs are only soluble under basic conditions and precipitate in the presence of divalent cations required for many downstream applications. Here, we show that the positively charged poly(styrene-co-maleimide) (SMI) forms similar nanoparticles with comparable efficiency to SMA, whilst remaining functional at acidic pH and compatible with high concentrations of divalent cations. We have performed a detailed characterization of the performance of SMI that enables a direct comparison with similar data published for SMA. We also demonstrate that SMI is capable of extracting proteins directly from the cell membrane and can solubilize functional human G-protein coupled receptors (GPCRs) expressed in cultured HEK 293T cells. “SMILPs” thus provide an alternative membrane solubilization method that successfully overcomes some of the limitations of the SMALP method.

Resumen: Las ciencias de la vida cubren una amplia gama de actividades relacionadas con los organismos vivos. Muchas de estas actividades se llevan a cabo en laboratorios de diagnóstico o investigación u otras instalaciones especializadas, como instalaciones de alojamiento de animales, unidades de cuarentena, invernaderos o instalaciones de producción biológica. Y muchas de estas actividades implican el uso de organismos y otros materiales biológicos que pueden presentar un peligro para las personas que los manipulan, pero también para la comunidad exterior o el medio ambiente, es decir, principalmente animales o plantas, en caso de escape y diseminación. La bioseguridad, y más ampliamente la gestión del riesgo biológico, tiene como objetivo prevenir, limitar o, en términos más generales, controlar estos riesgos. El enfoque de gestión del riesgo biológico se basa en una serie de medidas que involucran prácticas laborales, protección personal, infraestructura de contención y prácticas de gestión. Los propósitos de este primer capítulo son explicar las principales nociones y principios que los arquitectos e ingenieros que puedan trabajar en este campo en particular deben conocer y comprender, y presentar las principales referencias y puntos de vista adoptados en este folleto.

G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins and are an important target for therapeutic drugs. These receptors are highly dynamic proteins sampling a range of conformational states in order to fulfil their complex signalling roles. In order to fully understand GPCR signalling mechanisms it is necessary to extract the receptor protein out of the plasma membrane. Historically this has universally required detergents which inadvertently strip away the annulus of lipid in close association with the receptor and disrupt lateral pressure exerted by the bilayer. Detergent-solubilized GPCRs are very unstable which presents a serious hurdle to characterization by biophysical methods. A range of strategies have been developed to ameliorate the detrimental effect of removing the receptor from the membrane including amphipols and reconstitution into nanodics stabilized by membrane scaffolding proteins (MSPs) but they all require exposure to detergent. Poly(styrene-co-maleic acid) (SMA) incorporates into membranes and spontaneously forms nanoscale poly(styrene-co-maleic acid) lipid particles (SMALPs), effectively acting like a 'molecular pastry cutter' to 'solubilize' GPCRs in the complete absence of detergent at any stage and with preservation of the native annular lipid throughout the process. GPCR-SMALPs have similar pharmacological properties to membrane-bound receptor, exhibit enhanced stability compared with detergent-solubilized receptors and being non-proteinaceous in nature, are fully compatible with downstream biophysical analysis of the encapsulated GPCR.

G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins and are an important target for therapeutic drugs. These receptors are highly dynamic proteins sampling a range of conformational states in order to fulfil their complex signalling roles. In order to fully understand GPCR signalling mechanisms it is necessary to extract the receptor protein out of the plasma membrane. Historically this has universally required detergents which inadvertently strip away the annulus of lipid in close association with the receptor and disrupt lateral pressure exerted by the bilayer. Detergent-solubilized GPCRs are very unstable which presents a serious hurdle to characterization by biophysical methods. A range of strategies have been developed to ameliorate the detrimental effect of removing the receptor from the membrane including amphipols and reconstitution into nanodics stabilized by membrane scaffolding proteins (MSPs) but they all require exposure to detergent. Poly(styrene-co-maleic acid) (SMA) incorporates into membranes and spontaneously forms nanoscale poly(styrene-co-maleic acid) lipid particles (SMALPs), effectively acting like a 'molecular pastry cutter' to 'solubilize' GPCRs in the complete absence of detergent at any stage and with preservation of the native annular lipid throughout the process. GPCR-SMALPs have similar pharmacological properties to membrane-bound receptor, exhibit enhanced stability compared with detergent-solubilized receptors and being non-proteinaceous in nature, are fully compatible with downstream biophysical analysis of the encapsulated GPCR.

G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins and are an important target for therapeutic drugs. These receptors are highly dynamic proteins sampling a range of conformational states in order to fulfil their complex signalling roles. In order to fully understand GPCR signalling mechanisms it is necessary to extract the receptor protein out of the plasma membrane. Historically this has universally required detergents which inadvertently strip away the annulus of lipid in close association with the receptor and disrupt lateral pressure exerted by the bilayer. Detergent-solubilized GPCRs are very unstable which presents a serious hurdle to characterization by biophysical methods. A range of strategies have been developed to ameliorate the detrimental effect of removing the receptor from the membrane including amphipols and reconstitution into nanodics stabilized by membrane scaffolding proteins (MSPs) but they all require exposure to detergent. Poly(styrene-co-maleic acid) (SMA) incorporates into membranes and spontaneously forms nanoscale poly(styrene-co-maleic acid) lipid particles (SMALPs), effectively acting like a 'molecular pastry cutter' to 'solubilize' GPCRs in the complete absence of detergent at any stage and with preservation of the native annular lipid throughout the process. GPCR-SMALPs have similar pharmacological properties to membrane-bound receptor, exhibit enhanced stability compared with detergent-solubilized receptors and being non-proteinaceous in nature, are fully compatible with downstream biophysical analysis of the encapsulated GPCR.

G-protein-coupled receptors (GPCRs) form the largest class of membrane proteins and are an important target for therapeutic drugs. These receptors are highly dynamic proteins sampling a range of conformational states in order to fulfil their complex signalling roles. In order to fully understand GPCR signalling mechanisms it is necessary to extract the receptor protein out of the plasma membrane. Historically this has universally required detergents which inadvertently strip away the annulus of lipid in close association with the receptor and disrupt lateral pressure exerted by the bilayer. Detergent-solubilized GPCRs are very unstable which presents a serious hurdle to characterization by biophysical methods. A range of strategies have been developed to ameliorate the detrimental effect of removing the receptor from the membrane including amphipols and reconstitution into nanodics stabilized by membrane scaffolding proteins (MSPs) but they all require exposure to detergent. Poly(styrene-co-maleic acid) (SMA) incorporates into membranes and spontaneously forms nanoscale poly(styrene-co-maleic acid) lipid particles (SMALPs), effectively acting like a 'molecular pastry cutter' to 'solubilize' GPCRs in the complete absence of detergent at any stage and with preservation of the native annular lipid throughout the process. GPCR-SMALPs have similar pharmacological properties to membrane-bound receptor, exhibit enhanced stability compared with detergent-solubilized receptors and being non-proteinaceous in nature, are fully compatible with downstream biophysical analysis of the encapsulated GPCR.

G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR-SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([3H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.

G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR-SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR-SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([3H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.

In the past few years there has been a growth in the use of nanoparticles for stabilizing lipid membranes that contain embedded proteins. These bionanoparticles provide a solution to the challenging problem of membrane protein isolation by maintaining a lipid bilayer essential to protein integrity and activity. We have previously described the use of an amphipathic polymer (poly(styrene-co-maleic acid), SMA) to produce discoidal nanoparticles with a lipid bilayer core containing the embedded protein. However the structure of the nanoparticle itself has not yet been determined. This leaves a major gap in understanding how the SMA stabilizes the encapsulated bilayer and how the bilayer relates physically and structurally to an unencapsulated lipid bilayer. In this paper we address this issue by describing the structure of the SMA lipid particle (SMALP) using data from small angle neutron scattering (SANS), electron microscopy (EM), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC) and nuclear magnetic resonance spectroscopy (NMR). We show that the particle is disc shaped containing a polymer “bracelet” encircling the lipid bilayer. The structure and orientation of the individual components within the bilayer and polymer are determined showing that styrene moieties within SMA intercalate between the lipid acyl chains. The dimensions of the encapsulated bilayer are also determined and match those measured for a natural membrane. Taken together, the description of the structure of the SMALP forms the foundation for future development and applications of SMALPs in membrane protein production and analysis.[Figure not available: see fulltext.] © 2015, Tsinghua University Press and Springer-Verlag Berlin Heidelberg., SCOPUS: ar.j, info:eu-repo/semantics/published

In the past few years there has been a growth in the use of nanoparticles for stabilizing lipid membranes that contain embedded proteins. These bionanoparticles provide a solution to the challenging problem of membrane protein isolation by maintaining a lipid bilayer essential to protein integrity and activity. We have previously described the use of an amphipathic polymer (poly(styrene-co-maleic acid), SMA) to produce discoidal nanoparticles with a lipid bilayer core containing the embedded protein. However the structure of the nanoparticle itself has not yet been determined. This leaves a major gap in understanding how the SMA stabilizes the encapsulated bilayer and how the bilayer relates physically and structurally to an unencapsulated lipid bilayer. In this paper we address this issue by describing the structure of the SMA lipid particle (SMALP) using data from small angle neutron scattering (SANS), electron microscopy (EM), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC) and nuclear magnetic resonance spectroscopy (NMR). We show that the particle is disc shaped containing a polymer “bracelet” encircling the lipid bilayer. The structure and orientation of the individual components within the bilayer and polymer are determined showing that styrene moieties within SMA intercalate between the lipid acyl chains. The dimensions of the encapsulated bilayer are also determined and match those measured for a natural membrane. Taken together, the description of the structure of the SMALP forms the foundation for future development and applications of SMALPs in membrane protein production and analysis.[Figure not available: see fulltext.] © 2015, Tsinghua University Press and Springer-Verlag Berlin Heidelberg., SCOPUS: ar.j, info:eu-repo/semantics/published

The roles of extracellular residues of G-protein-coupled receptors (GPCRs) are not well defined compared with residues in transmembrane helices. Nevertheless, it has been established that extracellular domains of both peptide-GPCRs and amine-GPCRs incorporate functionally important residues. Extracellular loop 2 (ECL2) has attracted particular interest, because the x-ray structure of bovine rhodopsin revealed that ECL2 projects into the binding crevice within the transmembrane bundle. Our study provides the first comprehensive investigation into the role of the individual residues comprising the entire ECL2 domain of a small peptide-GPCR. Using the V1a vasopressin receptor, systematic substitution of all of the ECL2 residues by Ala generated 30 mutant receptors that were characterized pharmacologically. The majority of these mutant receptor constructs (24 in total) had essentially wild-type ligand binding and intracellular signaling characteristics, indicating that these residues are not critical for normal receptor function. However, four aromatic residues Phe189, Trp206, Phe209, and Tyr218 are important for agonist binding and receptor activation and are highly conserved throughout the neurohypophysial hormone subfamily of peptide-GPCRs. Located in the middle of ECL2, juxtaposed to the highly conserved disulfide bond, Trp206 and Phe209 project into the binding crevice. Indeed, Phe209 is part of the Cys-X-X-X-Ar (where Ar is an aromatic residue) motif, which is well conserved in both peptide-GPCRs and amine-GPCRs. In contrast, Phe189 and Tyr218, located at the extreme ends of ECL2, may be important for determining the position of the ECL2 cap over the binding crevice. This study provides mechanistic insight into the roles of highly conserved ECL2 residues.

The roles of extracellular residues of G-protein-coupled receptors (GPCRs) are not well defined compared with residues in transmembrane helices. Nevertheless, it has been established that extracellular domains of both peptide-GPCRs and amine-GPCRs incorporate functionally important residues. Extracellular loop 2 (ECL2) has attracted particular interest, because the x-ray structure of bovine rhodopsin revealed that ECL2 projects into the binding crevice within the transmembrane bundle. Our study provides the first comprehensive investigation into the role of the individual residues comprising the entire ECL2 domain of a small peptide-GPCR. Using the V1a vasopressin receptor, systematic substitution of all of the ECL2 residues by Ala generated 30 mutant receptors that were characterized pharmacologically. The majority of these mutant receptor constructs (24 in total) had essentially wild-type ligand binding and intracellular signaling characteristics, indicating that these residues are not critical for normal receptor function. However, four aromatic residues Phe189, Trp206, Phe209, and Tyr218 are important for agonist binding and receptor activation and are highly conserved throughout the neurohypophysial hormone subfamily of peptide-GPCRs. Located in the middle of ECL2, juxtaposed to the highly conserved disulfide bond, Trp206 and Phe209 project into the binding crevice. Indeed, Phe209 is part of the Cys-X-X-X-Ar (where Ar is an aromatic residue) motif, which is well conserved in both peptide-GPCRs and amine-GPCRs. In contrast, Phe189 and Tyr218, located at the extreme ends of ECL2, may be important for determining the position of the ECL2 cap over the binding crevice. This study provides mechanistic insight into the roles of highly conserved ECL2 residues.

The roles of extracellular residues of G-protein-coupled receptors (GPCRs) are not well defined compared with residues in transmembrane helices. Nevertheless, it has been established that extracellular domains of both peptide-GPCRs and amine-GPCRs incorporate functionally important residues. Extracellular loop 2 (ECL2) has attracted particular interest, because the x-ray structure of bovine rhodopsin revealed that ECL2 projects into the binding crevice within the transmembrane bundle. Our study provides the first comprehensive investigation into the role of the individual residues comprising the entire ECL2 domain of a small peptide-GPCR. Using the V1a vasopressin receptor, systematic substitution of all of the ECL2 residues by Ala generated 30 mutant receptors that were characterized pharmacologically. The majority of these mutant receptor constructs (24 in total) had essentially wild-type ligand binding and intracellular signaling characteristics, indicating that these residues are not critical for normal receptor function. However, four aromatic residues Phe189, Trp206, Phe209, and Tyr218 are important for agonist binding and receptor activation and are highly conserved throughout the neurohypophysial hormone subfamily of peptide-GPCRs. Located in the middle of ECL2, juxtaposed to the highly conserved disulfide bond, Trp206 and Phe209 project into the binding crevice. Indeed, Phe209 is part of the Cys-X-X-X-Ar (where Ar is an aromatic residue) motif, which is well conserved in both peptide-GPCRs and amine-GPCRs. In contrast, Phe189 and Tyr218, located at the extreme ends of ECL2, may be important for determining the position of the ECL2 cap over the binding crevice. This study provides mechanistic insight into the roles of highly conserved ECL2 residues.

The roles of extracellular residues of G-protein-coupled receptors (GPCRs) are not well defined compared with residues in transmembrane helices. Nevertheless, it has been established that extracellular domains of both peptide-GPCRs and amine-GPCRs incorporate functionally important residues. Extracellular loop 2 (ECL2) has attracted particular interest, because the x-ray structure of bovine rhodopsin revealed that ECL2 projects into the binding crevice within the transmembrane bundle. Our study provides the first comprehensive investigation into the role of the individual residues comprising the entire ECL2 domain of a small peptide-GPCR. Using the V1a vasopressin receptor, systematic substitution of all of the ECL2 residues by Ala generated 30 mutant receptors that were characterized pharmacologically. The majority of these mutant receptor constructs (24 in total) had essentially wild-type ligand binding and intracellular signaling characteristics, indicating that these residues are not critical for normal receptor function. However, four aromatic residues Phe189, Trp206, Phe209, and Tyr218 are important for agonist binding and receptor activation and are highly conserved throughout the neurohypophysial hormone subfamily of peptide-GPCRs. Located in the middle of ECL2, juxtaposed to the highly conserved disulfide bond, Trp206 and Phe209 project into the binding crevice. Indeed, Phe209 is part of the Cys-X-X-X-Ar (where Ar is an aromatic residue) motif, which is well conserved in both peptide-GPCRs and amine-GPCRs. In contrast, Phe189 and Tyr218, located at the extreme ends of ECL2, may be important for determining the position of the ECL2 cap over the binding crevice. This study provides mechanistic insight into the roles of highly conserved ECL2 residues.

For G-protein-coupled receptors (GPCRs) in general, the roles of extracellular residues are not well defined compared with residues in transmembrane helices (TMs). Nevertheless, extracellular residues are important for various functions in both peptide-GPCRs and amine-GPCRs. In this study, the V1a vasopressin receptor was used to systematically investigate the role of extracellular charged residues that are highly conserved throughout a subfamily of peptide-GPCRs, using a combination of mutagenesis and molecular modeling. Of the 13 conserved charged residues identified in the extracellular loops (ECLs), Arg116 (ECL1), Arg125 (top of TMIII), and Asp204 (ECL2) are important for agonist binding and/or receptor activation. Molecular modeling revealed that Arg125 (and Lys 125) stabilizes TMIII by interacting with lipid head groups. Charge reversal (Asp125) caused re-ordering of the lipids, altered helical packing, and increased solvent penetration of the TM bundle. Interestingly, a negative charge is excluded at this locus in peptide-GPCRs, whereas a positive charge is excluded in amine-GPCRs. This contrasting conserved charge may reflect differences in GPCR binding modes between peptides and amines, with amines needing to access a binding site crevice within the receptor TM bundle, whereas the binding site of peptide-GPCRs includes more extracellular domains. A conserved negative charge at residue 204 (ECL2), juxtaposed to the highly conserved disulfide bond, was essential for agonist binding and signaling. Asp204 (and Glu204) establishes TMIII contacts required for maintaining the α-hairpin fold of ECL2, which if broken (Ala204 or Arg204) resulted in ECL2 unfolding and receptor dysfunction. This study provides mechanistic insight into the roles of conserved extracellular residues.

For G-protein-coupled receptors (GPCRs) in general, the roles of extracellular residues are not well defined compared with residues in transmembrane helices (TMs). Nevertheless, extracellular residues are important for various functions in both peptide-GPCRs and amine-GPCRs. In this study, the V1a vasopressin receptor was used to systematically investigate the role of extracellular charged residues that are highly conserved throughout a subfamily of peptide-GPCRs, using a combination of mutagenesis and molecular modeling. Of the 13 conserved charged residues identified in the extracellular loops (ECLs), Arg116 (ECL1), Arg125 (top of TMIII), and Asp204 (ECL2) are important for agonist binding and/or receptor activation. Molecular modeling revealed that Arg125 (and Lys 125) stabilizes TMIII by interacting with lipid head groups. Charge reversal (Asp125) caused re-ordering of the lipids, altered helical packing, and increased solvent penetration of the TM bundle. Interestingly, a negative charge is excluded at this locus in peptide-GPCRs, whereas a positive charge is excluded in amine-GPCRs. This contrasting conserved charge may reflect differences in GPCR binding modes between peptides and amines, with amines needing to access a binding site crevice within the receptor TM bundle, whereas the binding site of peptide-GPCRs includes more extracellular domains. A conserved negative charge at residue 204 (ECL2), juxtaposed to the highly conserved disulfide bond, was essential for agonist binding and signaling. Asp204 (and Glu204) establishes TMIII contacts required for maintaining the α-hairpin fold of ECL2, which if broken (Ala204 or Arg204) resulted in ECL2 unfolding and receptor dysfunction. This study provides mechanistic insight into the roles of conserved extracellular residues.

The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of a family B G-protein-coupled receptor, calcitonin receptor-like receptor (CLR), and the accessory protein receptor activity modifying protein 1. It couples to Gs, but it is not known which intracellular loops mediate this. We have identified the boundaries of this loop based on the relative position and length of the juxtamembrane transmembrane regions 3 and 4. The loop has been analyzed by systematic mutagenesis of all residues to alanine, measuring cAMP accumulation, CGRP affinity, and receptor expression. Unlike rhodopsin, ICL2 of the CGRP receptor plays a part in the conformational switch after agonist interaction. His-216 and Lys-227 were essential for a functional CGRP-induced cAMP response. The effect of (H216A)CLR is due to a disruption to the cell surface transport or surface stability of the mutant receptor. In contrast, (K227A)CLR had wild-type expression and agonist affinity, suggesting a direct disruption to the downstream signal transduction mechanism of the CGRP receptor. Modeling suggests that the loop undergoes a significant shift in position during receptor activation, exposing a potential G-protein binding pocket. Lys-227 changes position to point into the pocket, potentially allowing it to interact with bound G-proteins. His-216 occupies a position similar to that of Tyr-136 in bovine rhodopsin, part of the DRY motif of the latter receptor. This is the first comprehensive analysis of an entire intracellular loop within the calcitonin family of G-protein-coupled receptor. These data help to define the structural and functional characteristics of the CGRP-receptor and of family B G-protein-coupled receptors in general. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of a family B G-protein-coupled receptor, calcitonin receptor-like receptor (CLR), and the accessory protein receptor activity modifying protein 1. It couples to Gs, but it is not known which intracellular loops mediate this. We have identified the boundaries of this loop based on the relative position and length of the juxtamembrane transmembrane regions 3 and 4. The loop has been analyzed by systematic mutagenesis of all residues to alanine, measuring cAMP accumulation, CGRP affinity, and receptor expression. Unlike rhodopsin, ICL2 of the CGRP receptor plays a part in the conformational switch after agonist interaction. His-216 and Lys-227 were essential for a functional CGRP-induced cAMP response. The effect of (H216A)CLR is due to a disruption to the cell surface transport or surface stability of the mutant receptor. In contrast, (K227A)CLR had wild-type expression and agonist affinity, suggesting a direct disruption to the downstream signal transduction mechanism of the CGRP receptor. Modeling suggests that the loop undergoes a significant shift in position during receptor activation, exposing a potential G-protein binding pocket. Lys-227 changes position to point into the pocket, potentially allowing it to interact with bound G-proteins. His-216 occupies a position similar to that of Tyr-136 in bovine rhodopsin, part of the DRY motif of the latter receptor. This is the first comprehensive analysis of an entire intracellular loop within the calcitonin family of G-protein-coupled receptor. These data help to define the structural and functional characteristics of the CGRP-receptor and of family B G-protein-coupled receptors in general. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of a family B G-protein-coupled receptor, calcitonin receptor-like receptor (CLR), and the accessory protein receptor activity modifying protein 1. It couples to Gs, but it is not known which intracellular loops mediate this. We have identified the boundaries of this loop based on the relative position and length of the juxtamembrane transmembrane regions 3 and 4. The loop has been analyzed by systematic mutagenesis of all residues to alanine, measuring cAMP accumulation, CGRP affinity, and receptor expression. Unlike rhodopsin, ICL2 of the CGRP receptor plays a part in the conformational switch after agonist interaction. His-216 and Lys-227 were essential for a functional CGRP-induced cAMP response. The effect of (H216A)CLR is due to a disruption to the cell surface transport or surface stability of the mutant receptor. In contrast, (K227A)CLR had wild-type expression and agonist affinity, suggesting a direct disruption to the downstream signal transduction mechanism of the CGRP receptor. Modeling suggests that the loop undergoes a significant shift in position during receptor activation, exposing a potential G-protein binding pocket. Lys-227 changes position to point into the pocket, potentially allowing it to interact with bound G-proteins. His-216 occupies a position similar to that of Tyr-136 in bovine rhodopsin, part of the DRY motif of the latter receptor. This is the first comprehensive analysis of an entire intracellular loop within the calcitonin family of G-protein-coupled receptor. These data help to define the structural and functional characteristics of the CGRP-receptor and of family B G-protein-coupled receptors in general. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of a family B G-protein-coupled receptor, calcitonin receptor-like receptor (CLR), and the accessory protein receptor activity modifying protein 1. It couples to Gs, but it is not known which intracellular loops mediate this. We have identified the boundaries of this loop based on the relative position and length of the juxtamembrane transmembrane regions 3 and 4. The loop has been analyzed by systematic mutagenesis of all residues to alanine, measuring cAMP accumulation, CGRP affinity, and receptor expression. Unlike rhodopsin, ICL2 of the CGRP receptor plays a part in the conformational switch after agonist interaction. His-216 and Lys-227 were essential for a functional CGRP-induced cAMP response. The effect of (H216A)CLR is due to a disruption to the cell surface transport or surface stability of the mutant receptor. In contrast, (K227A)CLR had wild-type expression and agonist affinity, suggesting a direct disruption to the downstream signal transduction mechanism of the CGRP receptor. Modeling suggests that the loop undergoes a significant shift in position during receptor activation, exposing a potential G-protein binding pocket. Lys-227 changes position to point into the pocket, potentially allowing it to interact with bound G-proteins. His-216 occupies a position similar to that of Tyr-136 in bovine rhodopsin, part of the DRY motif of the latter receptor. This is the first comprehensive analysis of an entire intracellular loop within the calcitonin family of G-protein-coupled receptor. These data help to define the structural and functional characteristics of the CGRP-receptor and of family B G-protein-coupled receptors in general. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.