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"STRUCTURAL AND MOLECULAR MECHANISMS THAT DETERMINE THE D3 DOPAMINE RECEPTOR TOLERANCE PROPERTY"

by
Sara Gil-Mast
Interdisciplinary Biomedical Sciences Program
B.Sc. (Hons), Kings College London, U.K.



Thesis Advisor: Eldo Kuzhikandathil, Ph.D.
Associate Professor
Department of Pharmacology and Physiology

Wednesday, April 27, 2011
MSB H-609B, 11:00 A.M.


Abstract

The D3 dopamine receptor (D3R) exhibits two unusual phenomena, tolerance and slow response termination (SRT). Tolerance is defined as a reduction in signaling response upon a second exposure to agonist; it is not the result of internalization or a change in the binding affinity. SRT occurs when the agonist is removed and is seen as a slow turn off of the response. Neither of these phenomena is seen in the closely related D2 dopamine receptor (D2R). While earlier studies had shown the importance of the D3R intracellular loop 2 (IL2) and more specifically the residue C147 in the D3R tolerance phenomena, it was found that reciprocal substitution of the IL2 region of the D3R into the D2S receptor did not induce tolerance.
Further studies have shown that the induction of tolerance is associated with a change in the conformation of extracellularly exposed regions of the receptor and that an agonist which does not induce tolerance does not induce the same change. We now show that the non-tolerant D3C147K receptor does not show the same change in conformation on exposure to tolerance causing agonists as wild type D3R. This supports a role of distal regions of the receptor in altering the intracellular signaling properties.
We have also shown that D187 a residue in the extracellular loop 2 (ec2) is important in the induction of tolerance. The exchange of this residue for a non-polar amino acid prevents the development of tolerance to four different agonists which induce tolerance in the wild-type receptor. The same point mutation introduced into the background of the D3C147K however did not prevent tolerance in this receptor to some agonists. This result suggests that there are varied ways in which tolerance can be induced in D3R and that they are mutually exclusive. The introduction of the reciprocal mutation of A188D into the D2R did not induce tolerance suggesting that although the D187 residue is important for the induction of tolerance it is not sufficient. We suggest that the role of this residue maybe as part of an ionic lock in the D3R and that the partner residue is not present in the D2R.
We additionally show that the intracellular signaling of the D3R may change due to a G-protein switch during the development of tolerance. We provide data suggesting that Gi interferes with D3R signaling via its cognate G-protein partner Go. We also show data supporting a change in the molecular weight of the complex in which D3R are found following the development of tolerance. This change may be due to association of the D3R with another signaling protein or to a change in the oligomerization status of the D3R.


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