Oligomerization of the serotonin(1A) receptor in live cells: a time-resolved fluorescence anisotropy approach.

نویسندگان

  • Yamuna Devi Paila
  • Mamata Kombrabail
  • G Krishnamoorthy
  • Amitabha Chattopadhyay
چکیده

The serotonin(1A) receptor is a representative member of the G-protein coupled receptor (GPCR) superfamily and serves as an important target in the development of therapeutic agents for neuropsychiatric disorders. Oligomerization of GPCRs is an important contemporary issue since it is believed to be a crucial determinant for cellular signaling. In this work, we monitored the oligomerization status of the serotonin(1A) receptor tagged to enhanced yellow fluorescent protein (5-HT(1A)R-EYFP) in live cells utilizing time-resolved fluorescence anisotropy decay. We interpret the unresolved fast component of the observed anisotropy decay as fluorescence resonance energy transfer (FRET) between 5-HT(1A)R-EYFP molecules (homo-FRET). Homo-FRET enjoys certain advantages over hetero-FRET in the analysis of receptor oligomerization. Our results reveal the presence of constitutive oligomers of the serotonin(1A) receptor in live cells. We further show that the oligomerization status of the receptor is independent of ligand stimulation and sphingolipid depletion. Interestingly, acute (but not chronic) cholesterol depletion appears to enhance the oligomerization process. Importantly, our results are independent of receptor expression level, thereby ruling out complications arising due to high expression. These results have potential implications in future therapeutic strategies in pathophysiological conditions in which serotonin(1A) receptors are implicated.

برای دانلود رایگان متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Organization of higher-order oligomers of the serotonin₁(A) receptor explored utilizing homo-FRET in live cells.

The serotonin₁(A) receptor is a representative member of the GPCR superfamily and serves as an important drug target. The possible role of GPCR oligomerization in receptor function is an active area of research. We monitored the oligomerization state of serotonin₁(A) receptors using homo-FRET and fluorescence lifetime measurements. Homo-FRET is estimated by a reduction in fluorescence anisotrop...

متن کامل

Fixation alters fluorescence lifetime and anisotropy of cells expressing EYFP-tagged serotonin1A receptor.

Fluorescence microscopic approaches represent powerful techniques to monitor molecular interactions in the cellular milieu. Measurements of fluorescence lifetime and anisotropy enjoy considerable popularity in this context. These measurements are often performed on live as well as fixed cells. We report here that formaldehyde-induced cell fixation introduces heterogeneities in the fluorescence ...

متن کامل

Time-resolved fluorescence anisotropy imaging applied to live cells.

We have developed a wide-field time-resolved imaging system to image quantitatively both the fluorescence lifetime and the rotational correlation time of a fluorophore. Using a polarization-resolved imager, we simultaneously image orthogonal polarization components of the fluorescence emission onto a time-gated intensified CCD. We demonstrate imaging of solvent viscosity variations through the ...

متن کامل

Determination of hemolysis, osmotic fragility and fluorescence anisotropy on irradiated red blood cells as a function of kV of medical diagnostic X-rays

Background: People occasionally undergo medical diagnostic X-ray examinations and expose their red blood cells to radiation. Radiation that is generated from medical diagnostic X-ray machines is widely used in medical diagnoses. One of the important parameters is kilo-voltage (kV) that is applied across the X-ray tube in medical diagnostic X-ray machines. Kilo-voltage influences the radiation d...

متن کامل

Membrane organization of the human serotonin(1A) receptor monitored by detergent insolubility using GFP fluorescence.

Insolubility in non-ionic detergents such as Triton X-100 at low temperature is a widely used biochemical criterion for characterization of membrane domains. In view of the emerging role of membrane organization in the function of G-protein coupled receptors, we have examined detergent insolubility of the 5-HT(1A) receptor in CHO cells using a novel GFP fluorescence approach developed by us. Us...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

عنوان ژورنال:
  • The journal of physical chemistry. B

دوره 115 39  شماره 

صفحات  -

تاریخ انتشار 2011