The Functional Significance of G Protein-Coupled Receptor Dimerization.
dc.contributor.author | Whorton, Matthew R. | en_US |
dc.date.accessioned | 2008-08-25T20:54:56Z | |
dc.date.available | NO_RESTRICTION | en_US |
dc.date.available | 2008-08-25T20:54:56Z | |
dc.date.issued | 2008 | en_US |
dc.date.submitted | en_US | |
dc.identifier.uri | https://hdl.handle.net/2027.42/60782 | |
dc.description.abstract | G protein-coupled receptors (GPCRs) are seven transmembrane domain proteins that transduce a diverse array extracellular signals across the plasma membrane and couple to the heterotrimeric family of G proteins. Like most intrinsic membrane proteins, GPCRs are capable of oligomerization and this has led to speculation that GPCR dimers may be required for receptor function and efficient activation of G proteins. One challenge in understanding the function of oligomers relates to the inability to separate monomeric and oligomeric receptor complexes in membrane environments using traditional biochemical approaches. In this thesis, I use a novel reconstitution technique based on high density lipoproteins (HDL) to circumvent this limitation. HDL particles are 10 nm diameter phospholipid bilayer discs surrounded by a dimer of the amphipathic protein apolipoprotein A-I. I first demonstrate that a prototypical GPCR, the β2 adrenergic receptor (β2AR), can be incorporated into the phospholipid bilayer of a reconstituted HDL (rHDL) particle together with the stimulatory heterotrimeric G protein, Gs. Single-molecule fluorescence imaging and fluorescence resonance energy transfer (FRET) analyses demonstrate that a single β2AR is incorporated per rHDL particle. The monomeric β2AR efficiently activates Gs and displays GTPγS-sensitive allosteric ligand-binding properties. I also demonstrate that another prototypical GPCR, rhodopsin, is monomeric and functional when incorporated into rHDL particles. The photoreceptor, rhodopsin, has been shown to exist as arrays of dimers in native tissues and thus provides an ideal system for directly comparing the function of monomers and oligomers. Monomeric rhodopsin•rHDL maintains the appropriate spectral properties with respect to photoactivation and formation of the active form, metarhodopsin II. Additionally, the kinetics of metarhodopsin II decay is similar between oligomeric rhodopsin in native membranes and monomeric rhodopsin in rHDL particles. Furthermore, photoactivation of monomeric rhodopsin•rHDL also results in the rapid activation of transducin, at a rate that is comparable to that found in native rod outer segments and 20-fold faster than rhodopsin in detergent micelles. Together, these data suggest that a monomeric receptor in a lipid bilayer is the minimal functional unit necessary for signaling, and that oligomerization is not an absolute requirement for this process. | en_US |
dc.format.extent | 3699848 bytes | |
dc.format.extent | 1373 bytes | |
dc.format.mimetype | application/pdf | |
dc.format.mimetype | text/plain | |
dc.language.iso | en_US | en_US |
dc.subject | G Protein-coupled Receptor | en_US |
dc.subject | Adrenergic | en_US |
dc.subject | Rhodopsin | en_US |
dc.subject | G Protein | en_US |
dc.subject | Dimerization | en_US |
dc.subject | High Density Lipoprotein | en_US |
dc.title | The Functional Significance of G Protein-Coupled Receptor Dimerization. | en_US |
dc.type | Thesis | en_US |
dc.description.thesisdegreename | PhD | en_US |
dc.description.thesisdegreediscipline | Pharmacology | en_US |
dc.description.thesisdegreegrantor | University of Michigan, Horace H. Rackham School of Graduate Studies | en_US |
dc.contributor.committeemember | Sunahara, Roger | en_US |
dc.contributor.committeemember | Iniguez-Lluhi, Jorge | en_US |
dc.contributor.committeemember | Linderman, Jennifer J. | en_US |
dc.contributor.committeemember | Neubig, Richard Robert | en_US |
dc.contributor.committeemember | Pratt, William B. | en_US |
dc.contributor.committeemember | Tesmer, John | en_US |
dc.subject.hlbsecondlevel | Pharmacy and Pharmacology | en_US |
dc.subject.hlbtoplevel | Health Sciences | en_US |
dc.description.bitstreamurl | http://deepblue.lib.umich.edu/bitstream/2027.42/60782/1/mwhorton_1.pdf | |
dc.owningcollname | Dissertations and Theses (Ph.D. and Master's) |
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