The adenylate cyclase-coupled vasopressin V2-receptor is highly laterally mobile in membranes of LLC-PK1 renal epithelial cells at physiological temperature.
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 beta-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 degrees C: the mobile fraction (f) was approximately 0.9, and the apparent lateral diffusion coefficient (D) approximately 3.0 X 10(-10) cm2/s. V2-receptor mobility greatly decreased with decreasing temperature: at 10 degrees C f was reduced to approximately 0.1.(ABSTRACT TRUNCATED AT 250 WORDS)
Jans
D A
DA
Max-Planck-Institut für Biophysik, Frankfurt am Main, FRG.
Peters
R
R
Zsigo
J
J
Fahrenholz
F
F
D000262:Adenylyl Cyclases / Q000502:physiology; D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D005456:Fluorescent Dyes / Q000378:metabolism / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D011945:Receptors, Angiotensin / Q000378:metabolism*; D017483:Receptors, Vasopressin; D013696:Temperature; D014667:Vasopressins / Q000378:metabolism*
D005456:Fluorescent Dyes; D011945:Receptors, Angiotensin; D017483:Receptors, Vasopressin; D014667:Vasopressins; D011494:Protein Kinases; D000262:Adenylyl Cyclases