Abstract
The lateral mobility of membrane-associated hormone receptors has been proposed to play an important role in signal transduction. Direct measurements, however, have shown that the receptors for insulin, epidermal growth factor and β-adrenergic antagonists exhibit low mobility at physiological temperature. The present study, which represents the first report of lateral mobility of a polypeptide hormone receptor coupled to adenylate cyclase, yielded quite different results. The lateral mobility of the vasopressin renal-type (V2)-receptor was measured in the basal plasma membrane of cells of the LLC-PK1 porcine epithelial line, using the technique of fluorescence microphotolysis (photobleaching) and a rhodamine-labelled analogue of vasopressin. The analogue, 1-deamino[8-lysine(N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP) was synthesized and shown to have binding properties and biological activities very similar to those of Arg8-vasopressin (AVP). TR-LVP could be used to label specifically the V2-receptor of living LLC-PK1 cells, whereby LLC-PK1 cells incubated with TR-LVP in the presence of a 100-fold excess of AVP, or cells from the LLC-PK1 V2-receptor-deficient line M18 incubated with TR-LVP could be used as controls for non-specific binding. Using optical sectioning, specific receptor mobility could be measured both in the absence and presence of free TR-LVP. The V2-receptor was found to be largely mobile at 37°C: the mobile fraction (f) was ~0.9, and the apparent lateral diffusion coefficient (D) ~3.0 x 10-10 cm2/s. V2-receptor mobility greatly decreased with decreasing temperature: at 10°C f was reduced to ~0.1. Lipid probe mobility demonstrated that receptor immobility at 10°C was not attributable to a temperature-induced phase transition of the plasma membrane lipid bilayer.
| Original language | English |
|---|---|
| Pages (from-to) | 2481-2488 |
| Number of pages | 8 |
| Journal | EMBO Journal |
| Volume | 8 |
| Issue number | 9 |
| Publication status | Published - 1 Jan 1989 |
| Externally published | Yes |
Cite this
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The adenylate cyclase-coupled vasopressin V2-receptor is highly laterally mobile in membranes of LLC-PK1 renal epithelial cells at physiological temperature. / Jans, D. A.; Peters, R.; Zsigo, J.; Fahrenholz, F.
In: EMBO Journal, Vol. 8, No. 9, 01.01.1989, p. 2481-2488.Research output: Contribution to journal › Article › Research › peer-review
TY - JOUR
T1 - The adenylate cyclase-coupled vasopressin V2-receptor is highly laterally mobile in membranes of LLC-PK1 renal epithelial cells at physiological temperature
AU - Jans, D. A.
AU - Peters, R.
AU - Zsigo, J.
AU - Fahrenholz, F.
PY - 1989/1/1
Y1 - 1989/1/1
N2 - The lateral mobility of membrane-associated hormone receptors has been proposed to play an important role in signal transduction. Direct measurements, however, have shown that the receptors for insulin, epidermal growth factor and β-adrenergic antagonists exhibit low mobility at physiological temperature. The present study, which represents the first report of lateral mobility of a polypeptide hormone receptor coupled to adenylate cyclase, yielded quite different results. The lateral mobility of the vasopressin renal-type (V2)-receptor was measured in the basal plasma membrane of cells of the LLC-PK1 porcine epithelial line, using the technique of fluorescence microphotolysis (photobleaching) and a rhodamine-labelled analogue of vasopressin. The analogue, 1-deamino[8-lysine(N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP) was synthesized and shown to have binding properties and biological activities very similar to those of Arg8-vasopressin (AVP). TR-LVP could be used to label specifically the V2-receptor of living LLC-PK1 cells, whereby LLC-PK1 cells incubated with TR-LVP in the presence of a 100-fold excess of AVP, or cells from the LLC-PK1 V2-receptor-deficient line M18 incubated with TR-LVP could be used as controls for non-specific binding. Using optical sectioning, specific receptor mobility could be measured both in the absence and presence of free TR-LVP. The V2-receptor was found to be largely mobile at 37°C: the mobile fraction (f) was ~0.9, and the apparent lateral diffusion coefficient (D) ~3.0 x 10-10 cm2/s. V2-receptor mobility greatly decreased with decreasing temperature: at 10°C f was reduced to ~0.1. Lipid probe mobility demonstrated that receptor immobility at 10°C was not attributable to a temperature-induced phase transition of the plasma membrane lipid bilayer.
AB - The lateral mobility of membrane-associated hormone receptors has been proposed to play an important role in signal transduction. Direct measurements, however, have shown that the receptors for insulin, epidermal growth factor and β-adrenergic antagonists exhibit low mobility at physiological temperature. The present study, which represents the first report of lateral mobility of a polypeptide hormone receptor coupled to adenylate cyclase, yielded quite different results. The lateral mobility of the vasopressin renal-type (V2)-receptor was measured in the basal plasma membrane of cells of the LLC-PK1 porcine epithelial line, using the technique of fluorescence microphotolysis (photobleaching) and a rhodamine-labelled analogue of vasopressin. The analogue, 1-deamino[8-lysine(N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP) was synthesized and shown to have binding properties and biological activities very similar to those of Arg8-vasopressin (AVP). TR-LVP could be used to label specifically the V2-receptor of living LLC-PK1 cells, whereby LLC-PK1 cells incubated with TR-LVP in the presence of a 100-fold excess of AVP, or cells from the LLC-PK1 V2-receptor-deficient line M18 incubated with TR-LVP could be used as controls for non-specific binding. Using optical sectioning, specific receptor mobility could be measured both in the absence and presence of free TR-LVP. The V2-receptor was found to be largely mobile at 37°C: the mobile fraction (f) was ~0.9, and the apparent lateral diffusion coefficient (D) ~3.0 x 10-10 cm2/s. V2-receptor mobility greatly decreased with decreasing temperature: at 10°C f was reduced to ~0.1. Lipid probe mobility demonstrated that receptor immobility at 10°C was not attributable to a temperature-induced phase transition of the plasma membrane lipid bilayer.
UR - http://www.scopus.com/inward/record.url?scp=0024456622&partnerID=8YFLogxK
M3 - Article
C2 - 2531084
AN - SCOPUS:0024456622
VL - 8
SP - 2481
EP - 2488
JO - EMBO Journal
JF - EMBO Journal
SN - 0261-4189
IS - 9
ER -