Although a long standing convention maintained that G Protein Coupled Receptors (GPCRs) exist in the plasma membrane solely as monomers, substantial work over the last two decades has demonstrated that these ubiquitous receptors can and in many cases, preferentially, exist as homodimers, heterodimers, or higher order oligomers. Often, two GPCRs of the same class heterodimerize; it is less common for two GPCRs of different signaling pathways to interact. The work presented here studied the physical and functional interaction of two GPCRs from discrete classes, the Beta 2 Adrenergic Receptor (β2AR), a Gαs-coupled receptor, and Bradykinin type 2 Receptor (Bk2R), a Gαq coupled receptor. These data show that Bk2R and β2AR are physically coupled when heterologously expressed in Xenopus oocytes, and in pheochromocytoma (PC12) cells and in freshly isolated murine ventricular myocytes, two systems that endogenously express these receptors. This physical coupling led to functional consequences in heterologous and endogenous expression systems, as Bk2R was able to transactivate β2AR signaling via its direct interaction with the receptor. Furthermore, coexpression of Bk2R shifted the dose response curve of β2AR for its selective agonist rightward in Xenopus oocyte electrophysiology experiments, suggesting the presence of Bk2R negatively affected β2AR native pharmacology. Up to thirty minutes of either bradykinin (BK) or isoproterenol exposure did not change the relative amount of Bk2R/β2AR heterodimer in PC12 cells, a rat adrenal medulla tumor cell line that endogenously expresses these receptors. Despite the obvious signaling consequences, the Bk2R/β2AR heterodimer accounted for only 10% of the total β2AR protein detected and 20% of the total Bk2R protein detected. When other Bk2R-specific ligands were also tested to examine the extent of β2AR transactivation, our data showed that both Lys-des-Arg-Bradykinin, a Bk2R partial agonist and NPC 567, a Bk2R antagonist, transactivated β2AR to the same extent as BK. Taken together, our data provide a novel mode of receptor regulation and signaling via Bk2R/β2AR heterodimerization. Because a large percentage of therapeutics target GPCRs, a greater understanding of how a GPCR heterodimer functions could be beneficial for targeting new drugs and refining existing drugs. Understanding the Bk2R/β2AR heterodimer provides a new perspective on the myriad of fucntional consequences that occur when a GPCR is activated.

Trace amines (TAs), tryptamine, tyramine, octopamine, and beta-phenylethylamine, named for their low endogenous concentrations in mammals, are related to the classical monoamine transmitters, but have been understudied and thought of as false transmitters. They share structural, physiological, pharmacological, and metabolic similarities with the monoamines, including synthesis by the aromatic-L-amino acid decarboxylase (AADC) enzyme. In 2001, a new class of receptors preferentially activated by the TAs, termed trace amine-associated receptors (TAARs), was discovered establishing a mechanism for TA actions independent of classic monoaminergic mechanisms. While the TAs and some of their receptors are present in the mammalian central nervous system (CNS), their physiologic role remains uncertain. I hypothesized that the TAs are found intrinsically in the spinal cord, and that they are able to modulate spinal neural networks. Using immunohistochemistry, numerous spinal neurons were identified that express AADC, TAs, and TAARs. Similar results were seen for AADC and TAAR1 with in situ hybridization. The most consistent observation was for labeling D cells associated with the central canal and in motoneurons. Overall, these results provided evidence for the presence of an anatomical substrate onto which the TAs could have intrinsic biological actions in the spinal cord. Using exogenous application of the TAs in the isolated spinal cord in vitro, and in vivo in the mid-thoracic chronically spinalized, I showed that the TAs could induce rhythmic locomotor-like activity. TA injection-induced hindlimb motor rhythms observed in chronic spinalized animals, supports TA spinal actions independent of the descending monoaminergic systems. In the presence of NMDA, TA applications recruited a variety of rhythmic motor patterns in the isolated spinal cord. This ranged from locomotor activity indistinguishable from 5-HT/NMDA induced locomotion to complex patterns including, an episodic form of locomotion where there were locomotor bouts with intervening quiescent periods. TA actions of pattern generating circuits had slower kinetics of activation than 5-HT and NA, were attenuated in the presence of monoamine transport inhibitors, and had increased intracellular labeling when incubated in a nominally Na+-free solution. Together these results suggest that the TAs require transport into neurons to exert their actions, and that transport occurs by Na+-dependent monoamine transporters as well as Na+-independent transporters. Finally, I used the in vitro isolated spinal cord with attached hindlimbs to record electromyographic (EMG) activity from various hindlimb muscles to compare the relationship between the TAs and serotonin (5-HT) evoked motor coordination and to examine the ability of the TAs to modulate ongoing 5-HT and NMDA locomotor-like activity. The TAs produced both the continuous and episodic patterns on muscles as observed in ventral root recordings, but EMG recordings provided more detailed insight into specific muscle actions. The TAs also generally increased both frequency and amplitude of ongoing 5-HT locomotor frequency, with tyramine and octopamine also particularly able to alter 5-HT motor coordination patterns.

Thesis (Ph. D.)--Florida State University, 2003. / Advisor: Dr. Paul Trombley, Florida State University, School of Arts and Sciences, Dept. of Biological Science. Title and description from dissertation home page (viewed Feb. 26, 2004). Includes bibliographical references.

Thesis (Ph.D.)--Ohio University, November, 2009. / Release of full electronic text on OhioLINK has been delayed until December 1, 2014. Title from PDF t.p. Includes bibliographical references.

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Biology." Includes bibliographical references (p. 107-114).

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 151-180). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--York University, 1997. Graduate Programme in Psychology. / Typescript. Includes bibliographical references (leaves 93-99). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL:http://wwwlib.umi.com/cr/yorku/fullcit?pNQ22895.

Thesis (Ph. D.)--University of California, San Diego, 2003. / Vita. Includes bibliographical references (leaves 297-313).

Thesis--Ph. D., University of Hong Kong. / Includes reprints of 4 papers by the author and others in the appendices.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2003. / Vita. Includes bibliographical references.