whisper
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Agonist-induced Signaling and Endocytosis of the Adenosine A2A Receptor
Frank Brand
Abstract:
Adenosine, a nucleoside composed of adenine attached to a ribose, is an essential compound of life,
acting among others in cellular energy budget, inheritance, and regulating many bodily functions.
During the last decades, the study of the G protein-coupled adenosine receptor family resulted in many
discoveries improving human health. One of the four known adenosine receptor subtypes, referred to
as A1, A2A, A2B, and A3, is the focus of this thesis.
The Gs-coupled adenosine A2A receptor (A2AR), which is mainly responsible for the physiological
effect of the most common used drug caffeine, is still a major target for future drug development. An
important aspect for the understanding of adenosine receptor physiology is the investigation of
receptor dynamics and redistribution following agonist exposure. Therefore, two A2AR-specific
fluorescent agonists, called FITC-APEC and Alexa488-APEC, were applied for receptor targeting and
analysis of agonist-induced receptor trafficking. The fluorescent agonists activated cellular A2AR
signaling, i.e. receptor stimulation led to an increase in 3'-5'-cyclic adenosine monophosphate (cAMP)
as well as phosphorylation of the cAMP response element binding protein (CREB) in Chinese hamster
ovary (CHO) cells expressing the human A2AR. Furthermore, Alexa488-APEC was used in
combination with a cyan fluorescent protein (CFP)-tagged A2A receptor in suitable living cells to
investigate receptor binding and agonist-induced receptor trafficking. Alexa488-APEC binding
overlapped with the CFP-A2AR confirming receptor binding and specificity. Moreover, Alexa488-
APEC induced a time- and concentration-dependent receptor internalization, which could be blocked
by hyperosmolaric sucrose as well as a competitive antagonist ZM 241,385. Finally, the interaction of
major elements in G protein-coupled receptor (GPCR) endocytosis such as GPCR kinase 2 (GRK2), ßarrestins,
clathrin, and the small GTPase Rab5 could be visualized in living or fixed cells and related
to A2A receptor-redistribution.
In summary, it was shown that it is possible to detect the A2AR using the two fluorescent agonist.
Especially, Alexa488-APEC is able to specifically activate the A2AR signal transduction in nanomolar
concentrations and also promotes internalization of A2AR. In addition, GRK2, ß-arrestin1/2, clathrin,
and Rab5 are involved in A2AR-trafficking. For the future, it will be challenging to optimize the
agonists Alexa488/FITC-APEC for tissue-specific A2AR staining, which is of interest, for example, for
in vivo studies of the receptor dynamics in primary sensory neurons and its role in pain perception.
Pages:74
Frank Brand
Abstract:
Adenosine, a nucleoside composed of adenine attached to a ribose, is an essential compound of life,
acting among others in cellular energy budget, inheritance, and regulating many bodily functions.
During the last decades, the study of the G protein-coupled adenosine receptor family resulted in many
discoveries improving human health. One of the four known adenosine receptor subtypes, referred to
as A1, A2A, A2B, and A3, is the focus of this thesis.
The Gs-coupled adenosine A2A receptor (A2AR), which is mainly responsible for the physiological
effect of the most common used drug caffeine, is still a major target for future drug development. An
important aspect for the understanding of adenosine receptor physiology is the investigation of
receptor dynamics and redistribution following agonist exposure. Therefore, two A2AR-specific
fluorescent agonists, called FITC-APEC and Alexa488-APEC, were applied for receptor targeting and
analysis of agonist-induced receptor trafficking. The fluorescent agonists activated cellular A2AR
signaling, i.e. receptor stimulation led to an increase in 3'-5'-cyclic adenosine monophosphate (cAMP)
as well as phosphorylation of the cAMP response element binding protein (CREB) in Chinese hamster
ovary (CHO) cells expressing the human A2AR. Furthermore, Alexa488-APEC was used in
combination with a cyan fluorescent protein (CFP)-tagged A2A receptor in suitable living cells to
investigate receptor binding and agonist-induced receptor trafficking. Alexa488-APEC binding
overlapped with the CFP-A2AR confirming receptor binding and specificity. Moreover, Alexa488-
APEC induced a time- and concentration-dependent receptor internalization, which could be blocked
by hyperosmolaric sucrose as well as a competitive antagonist ZM 241,385. Finally, the interaction of
major elements in G protein-coupled receptor (GPCR) endocytosis such as GPCR kinase 2 (GRK2), ßarrestins,
clathrin, and the small GTPase Rab5 could be visualized in living or fixed cells and related
to A2A receptor-redistribution.
In summary, it was shown that it is possible to detect the A2AR using the two fluorescent agonist.
Especially, Alexa488-APEC is able to specifically activate the A2AR signal transduction in nanomolar
concentrations and also promotes internalization of A2AR. In addition, GRK2, ß-arrestin1/2, clathrin,
and Rab5 are involved in A2AR-trafficking. For the future, it will be challenging to optimize the
agonists Alexa488/FITC-APEC for tissue-specific A2AR staining, which is of interest, for example, for
in vivo studies of the receptor dynamics in primary sensory neurons and its role in pain perception.
Pages:74
[thanks-thanks]pdf,2.96MB, http://depositfiles.com/files/pob3myqjc[/thanks-thanks]
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