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Specific oligomerization of the 5-HT1A receptor in the plasma membrane.

Woehler A, Wlodarczyk J, Ponimaskin EG - Glycoconj. J. (2008)

Bottom Line: Furthermore we study the role of acylation in this interaction through measurements of a palmitoylation-deficient 5-HT(1A) receptor mutant.Palmitoylation increases the tendency of a receptor to localize in lipid rich microdomains of the plasma membrane.This increases the effective surface density of the receptor and provides for a higher level of stochastic interaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuro and Sensory Physiology, University of Göttingen, Gottingen, Germany.

ABSTRACT
In the present study we analyze the oligomerization of the 5-HT1A receptor within living cells at the sub-cellular level. Using a 2-excitation Förster Resonance Energy Transfer (FRET) method combined with spectral microscopy we are able to estimate the efficiency of energy transfer based on donor quenching as well as acceptor sensitization between CFP-and YFP-tagged 5-HT1A receptors at the plasma membrane. Through the analysis of the level of apparent FRET efficiency over the various relative amounts of donor and acceptor, as well as over a range of total surface expressions of the receptor, we verify the specific interaction of these receptors. Furthermore we study the role of acylation in this interaction through measurements of a palmitoylation-deficient 5-HT(1A) receptor mutant. Palmitoylation increases the tendency of a receptor to localize in lipid rich microdomains of the plasma membrane. This increases the effective surface density of the receptor and provides for a higher level of stochastic interaction.

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Oligomeriztion of the palmitoylation-deficient 5-HT1A receptor measured by lux-FRET. (A) lux-FRET images of N1E-115 cells expressing palmitoylation-deficient mutant receptors show that the membrane distribution of the calculated values appear no different than for the wild-type receptors shown in Fig. 2A. (B) The characterization of FRET as a function of donor mole fraction further shows that there is little difference between the interaction of the wild type receptors and the interaction of the mutant receptors. Application of the previously mentioned models suggests that for the EfD data E = 0.21 and n = 1.70 (R2 = 0.86) and for the EfA data E = 0.17 and n = 2.10 (R2 = 0.89). (C) Similarly to the wild-type receptor, plotting EfA values against spatially corresponding concentration values shows that there is no dependence of measured FRET on surface concentration of the palmitoylation-deficient 5-HT1A receptor
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Fig3: Oligomeriztion of the palmitoylation-deficient 5-HT1A receptor measured by lux-FRET. (A) lux-FRET images of N1E-115 cells expressing palmitoylation-deficient mutant receptors show that the membrane distribution of the calculated values appear no different than for the wild-type receptors shown in Fig. 2A. (B) The characterization of FRET as a function of donor mole fraction further shows that there is little difference between the interaction of the wild type receptors and the interaction of the mutant receptors. Application of the previously mentioned models suggests that for the EfD data E = 0.21 and n = 1.70 (R2 = 0.86) and for the EfA data E = 0.17 and n = 2.10 (R2 = 0.89). (C) Similarly to the wild-type receptor, plotting EfA values against spatially corresponding concentration values shows that there is no dependence of measured FRET on surface concentration of the palmitoylation-deficient 5-HT1A receptor

Mentions: To analyze the role of receptor palmitoylation for the oligomerization at the plasma membrane, we performed similar single-cell FRET analysis for the cells expressing CFP-and YFP-tagged acylation-deficient mutants (Fig. 3A). Comparison of the panel A in Figs. 2 and 3 does not reveal any significant differences in receptor or FRET distribution at the plasma membrane between wild-type and mutated receptors. These figures also demonstrate that the overall levels of the FRET for the cases of wild-type and non-palmitoylated receptor expression were comparable. However, we obtained a slight difference in the relationship of FRET to the donor mole fraction. In the case of the mutant receptor, the limiting value of EfD approaches the same value as EfA, while with the wild-type receptor the limit of the EfA value was greater than that of the EfD. Fitting the data according to the oligomerization model for the EfD produced the value of E = 0.21 and n = 1.70 (R2 = 0.8641) and for EfA-values of E = 0.17 and n = 2.10 (R2 = 0.8874) for the non-palmitoylated receptors.Fig 3


Specific oligomerization of the 5-HT1A receptor in the plasma membrane.

Woehler A, Wlodarczyk J, Ponimaskin EG - Glycoconj. J. (2008)

Oligomeriztion of the palmitoylation-deficient 5-HT1A receptor measured by lux-FRET. (A) lux-FRET images of N1E-115 cells expressing palmitoylation-deficient mutant receptors show that the membrane distribution of the calculated values appear no different than for the wild-type receptors shown in Fig. 2A. (B) The characterization of FRET as a function of donor mole fraction further shows that there is little difference between the interaction of the wild type receptors and the interaction of the mutant receptors. Application of the previously mentioned models suggests that for the EfD data E = 0.21 and n = 1.70 (R2 = 0.86) and for the EfA data E = 0.17 and n = 2.10 (R2 = 0.89). (C) Similarly to the wild-type receptor, plotting EfA values against spatially corresponding concentration values shows that there is no dependence of measured FRET on surface concentration of the palmitoylation-deficient 5-HT1A receptor
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2714455&req=5

Fig3: Oligomeriztion of the palmitoylation-deficient 5-HT1A receptor measured by lux-FRET. (A) lux-FRET images of N1E-115 cells expressing palmitoylation-deficient mutant receptors show that the membrane distribution of the calculated values appear no different than for the wild-type receptors shown in Fig. 2A. (B) The characterization of FRET as a function of donor mole fraction further shows that there is little difference between the interaction of the wild type receptors and the interaction of the mutant receptors. Application of the previously mentioned models suggests that for the EfD data E = 0.21 and n = 1.70 (R2 = 0.86) and for the EfA data E = 0.17 and n = 2.10 (R2 = 0.89). (C) Similarly to the wild-type receptor, plotting EfA values against spatially corresponding concentration values shows that there is no dependence of measured FRET on surface concentration of the palmitoylation-deficient 5-HT1A receptor
Mentions: To analyze the role of receptor palmitoylation for the oligomerization at the plasma membrane, we performed similar single-cell FRET analysis for the cells expressing CFP-and YFP-tagged acylation-deficient mutants (Fig. 3A). Comparison of the panel A in Figs. 2 and 3 does not reveal any significant differences in receptor or FRET distribution at the plasma membrane between wild-type and mutated receptors. These figures also demonstrate that the overall levels of the FRET for the cases of wild-type and non-palmitoylated receptor expression were comparable. However, we obtained a slight difference in the relationship of FRET to the donor mole fraction. In the case of the mutant receptor, the limiting value of EfD approaches the same value as EfA, while with the wild-type receptor the limit of the EfA value was greater than that of the EfD. Fitting the data according to the oligomerization model for the EfD produced the value of E = 0.21 and n = 1.70 (R2 = 0.8641) and for EfA-values of E = 0.17 and n = 2.10 (R2 = 0.8874) for the non-palmitoylated receptors.Fig 3

Bottom Line: Furthermore we study the role of acylation in this interaction through measurements of a palmitoylation-deficient 5-HT(1A) receptor mutant.Palmitoylation increases the tendency of a receptor to localize in lipid rich microdomains of the plasma membrane.This increases the effective surface density of the receptor and provides for a higher level of stochastic interaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuro and Sensory Physiology, University of Göttingen, Gottingen, Germany.

ABSTRACT
In the present study we analyze the oligomerization of the 5-HT1A receptor within living cells at the sub-cellular level. Using a 2-excitation Förster Resonance Energy Transfer (FRET) method combined with spectral microscopy we are able to estimate the efficiency of energy transfer based on donor quenching as well as acceptor sensitization between CFP-and YFP-tagged 5-HT1A receptors at the plasma membrane. Through the analysis of the level of apparent FRET efficiency over the various relative amounts of donor and acceptor, as well as over a range of total surface expressions of the receptor, we verify the specific interaction of these receptors. Furthermore we study the role of acylation in this interaction through measurements of a palmitoylation-deficient 5-HT(1A) receptor mutant. Palmitoylation increases the tendency of a receptor to localize in lipid rich microdomains of the plasma membrane. This increases the effective surface density of the receptor and provides for a higher level of stochastic interaction.

Show MeSH
Related in: MedlinePlus