Year of Award

2008

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Pharmaceutical Sciences

Other Degree Name/Area of Focus

Pharmacology

Department or School/College

College of Health Professions and Biomedical Sciences

Committee Chair

Keith K. Parker

Commitee Members

Jerry Smith, Kathleen George, John Gerdes, Jean Carter

Keywords

5HT, cAMP, G Protein Coupled Receptors, Peptides, Serotonin, Thermodynamics

Abstract

Hall, Brian, Ph.D., May 2008 Pharmacology/Pharmaceutical Sciences

Structure Activity Relationships for Intracellular Loop 2 of the 5HT1A Serotonin Receptor

Chairperson: Dr. Keith Parker

The human (H) serotonin (5-hydroxytrptamine; 5HT) 1a receptor (R) has been implicated in various physiological processes such as mood regulation, vascular and temperature control, anxiety, depression, and migraine headache. This seven transmembrane domain (7TMD), G protein-coupled receptor (GPCR) is negatively coupled to adenylyl cyclase (AC). This work was designed to better understand the coupling and activation requirements of intracellular loop 2 (ic2) with Gi in Chinese Hamster Ovary (CHO) cells. 10 MER peptides that are derived from the known sequence of the cloned receptor have been used as probes and the current study includes peptides P21 to P27 of ic2 (LDRYWAITDPIDYVNKRTPRPR), approaching the ic2 carboxy terminus of TMD4 two residues per 10 MER. Peptide ability to uncouple receptor from G protein was determined in agonist inhibition studies using the selective 5HT1aR agonist [3H]8-OH-DPAT. Peptides P21 to P23 uncoupled the G protein from the receptor to 50% of control. Peptides P24 to P27 uncoupled the G protein from the receptor to 6-15% of control. Peptide capacity to alter signal transduction was measured using enzyme-linked assays of intracellular cyclic AMP (cAMP) and assays quantifying incorporation of [35S]-?-Guanosine Triphosphate (GTP). Peptides closer to TMD3, but in the C-terminal aspect of ic2, were capable of uncoupling the receptor-G protein, suggesting a role for that region of the native receptor in G protein coupling (and activation). The final peptides in this region, P25 to P27 are relatively inactive with respect to G protein coupling and activation. Thus, we propose that the immediate ic2/TMD4 interface is beyond the receptor segment involved in coupling. Peptides P22-P24 were the most active with respect to stimulation of [35S]-?-GTP incorporation and decreasing cAMP concentration, therefore they were chosen for a thermodynamic binding study. The Kd for [3H]8-OH-DPAT was measured over a range of temperatures, which allowed the calculation of Standard Gibb's Free energy (?G∞), enthalpy (?H∞), and entropy (?S∞). The peptides increased the enthalpy and entropy of the system. These results contribute to structural information about 5HT1aR's interaction with Gi derived from a number of different, but compatible techniques. The developing model for ic2 (and ic3) loop-G protein regulation has implications for developing new therapeutic drugs for serotonergic pathologies such as the affective disorders.

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© Copyright 2008 Brian Patrick Hall