G Protein Coupled Receptors

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• G Protein Coupled Receptors

A project of David Lutje Hulsik and Tim Hulsen
May 7th, 2001
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What are GPCRs?

• Membrane-bound receptors

• Transducing messages as photons, organic
  odorants, nucleotides, nucleosides, peptides,
  lipids and proteins.

• 6 different families

• A very large number of different domains both to
  bind their ligand and to activate G proteins.

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GPCR Structure

• Seven transmembrane regions

• Hydrophobic/ hydrophilic domains

• Conserved residues and motifs (i.e. NPXXY)

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GPCR-G protein coupling

• Receptor gets activated by agonist

• G protein binds to activated receptor

• Agonist binding to receptor becomes stronger
  upon G protein coupling

• GDP is released

• G protein takes up GTP

• GTP uptake triggers release of G protein from
  receptor

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Research goals

• To determine whether predictions made about the
  structure of GPCRs are correct

• To see which methods give the best results

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Major research difficulties

• Residue numbering Schwartz / Baldwin
• (e.g. V.16)
• Ballesteros-Weinstein
• (e.g. 6.50)
• etc.

• Available high-resolution structural information

• Bacteriorhodopsin as template

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Bacteriorhodopsin

• Photosynthetic bacteria

• Proton pumping

• G proteins not involved

• Helical arrangement

• Conformation

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Studies on GPCRs

• Mutation studies

• Spinlabel

• NMR

• Cystein scanning

• Protease studies

• Photoaffinity

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Methods

• Collecting data

• Making alignment with the use of several
  articles
• which compare a GPCR with bacteriorhodopsin

• Structure validation with WHAT-IF

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Collecting Data

• Articles
• Oldfashioned library work
• Online libraries (PubMed)

• Websites
• Different GPCR-groups

• Online databases
• GPCRDB

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Making Alignments

• Structural alignment rhodopsin/bacteriorhodopsin
• ---------- -WIWLALGTA LMGLGTLYFL VK-------- BRh
• ----PWQFSM LAAYMFLLIM LGFPINFLTL YVTVQ----- Rh
• Comparing with alignments made by other
  GPCR-experts
• ---------- -WIWLALGTA LMGLGTLYFL VK-------- BRh
• ----PWQFSM LAAYMFLLIM LGFPINFLTL YVTVQ----- Rh
• ---------P EWIWLALGTA LMGLGTLYFL VKGM------ BRh
  Vriend
• ---------Q FSMLAAYMFL LIMLGFPINF LTLY------ Rh
  Vriend
• Difference 3
• Helix 3 means trouble
• Differences were larger then 10.

Complete alignment
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Structure validation with WHAT-IF I

• Structure predictions by Baldwin et al.
• Electron density maps
• 493 GPCR (a.a.)sequences
• Helical orientation
• Interacting residues

Helical orientation as predicted was correct Only
a few residues interact 17 G P VII18 18 N A
II11 D II14 21 V A II11 Y VII21
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Structure validation with WHAT-IF II

• Structure predictions by Thirstrup et al.
• Construction of zinc binding site
• ?-opioid receptor
• Helical orientation
• Helix-helix interactions

Measured distances between zinc ion and residues
too large even with the use of the tors command
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Structure validation with WHAT-IF III

• Structure predictions by Greenhalgh et al.
• Spin label electron paramagnetic resonance
  spectroscopy
• Mapping residue positions
• (relative to aqueous boundaries)

Distances for bacteriorhodopsin (in Å)

Differences are within range spin-labeling could
be a reasonably safe way to predict the structure
of membrane proteins
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Conclusions

• Predicting a structure with such low level of
  homology is very hard

• Availability of real data (e.g. electron density
  maps) improves structure prediction

• Most predictions are in the right direction, but
  still need some refinement

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Take a look at our website!
http//go.to/gpcr
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Learning points

• Programming in Python

• What-IF

• GPCRs

• Website building (e.g. CGI)

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