Influence of GPCR coexpression in neuronal cells on the convergence of signaling pathways

Influence of GPCR coexpression in neuronal cells on the convergence of signaling pathways

Tim Ullrich

Doctoral thesis

Date of Examination: 2013-07-29

Date of issue: 2013-07-18

Advisor: Salonikidis, Petrus S. Dr.

Referee: Richter, Diethelm Prof. Dr.

Referee: Nikolaev, Viacheslav Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-0001-BACC-3

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Abstract

English

Receptor crosstalk is a universal feature of signaling and interference happens on manifold pathway junctions. The physiological occurrence of the two serotonin receptor subtypes 5-HT1A and 5-HT7 in the same neuronal cells gave rise to the question of how their counter-cooperative impact merges on the mutual target adenylyl cyclase (AC). 5-HT1A receptors are generally known to decrease the cellular cAMP concentration via Gi mediated AC inhibition. In contrast, 5-HT7 receptors commonly increase [cAMP] via AC stimulating Gs proteins. Förster resonance energy transfer (FRET) measurements were performed in 5-HT1A – 5-HT7 coexpressing cells to track cAMP development during stimulation in real time. Fluorescent receptor labeling and visual selection of the measuring cells, combined with supporting FACS data and functional analysis ensured equal receptor expression in analyzed cells. An additional consideration is that many serotonin receptors, including 5-HT1A and 5-HT7, tend to oligomerize. Oligomerization has been shown to alter signaling in a vast number of cases and might play a key role in the 5-HT1A – 5-HT7 interaction. Coexpressing cells were stimulated with serotonin to examine what impact the common receptor expression had on [cAMP] and if one pathway appeared to be dominant. Subsequent specific inhibition of either receptor was supposed to review respective binding sites and further reveal the nature of 5-HT1A – 5-HT7 interaction. Blocking on the G protein level allowed analysis of the last step of the 5-HT pathway before the preliminary end target, AC. The key results of the actual thesis are summarized below: 1. Upon 5-HT1A – 5-HT7 coexpression a strong functional interaction with dominance of the Gi pathway became apparent. Coactivation of coexpressing cells leads to significant [cAMP] decrease. 2. Specific blockade of either receptor almost totally prevents 5-HT signaling in these cells. 3. Specific 5-HT7 stimulation likewise induces a [cAMP] decrease. Oligomerization of the two serotonin receptors might be a possible explanation for the newly observed signaling features. The available results cannot predict with certainty whether oligomerization occurs or not, but in any case a strong interaction became evident and gives rise to further investigation of serotonin receptor coexpression.

Keywords: 5-HT1A; 5-HT7; Receptor oligomerization; Receptor crosstalk; serotonin receptors