Receptor Watching Enabled

Abstract

Yudowski et al. report that they have been able to observe insertion of single receptor-containing vesicles into the plasma membrane of cultured hippocampal neurons from rats. The authors accomplished this by expressing in the cells a modified form of the β 2 -adrenergic receptor (β 2 AR) in which the extracellular end of the receptor was tagged with a variant of the green fluorescent protein (GFP). The fluorescence of this particular GFP protein is quenched when exposed to the acidic environment of endocytic vesicles but increases when exposed to culture medium of pH 7.4 at the cell surface. The vesicle fusion events appeared to represent recycling of internalized receptors to the cell surface because they were detected only after exposure of the cells to the β 2 AR agonist isoproterenol (which causes receptor internalization and subsequent reinsertion in the plasma membrane). Two distinct modes of insertion were detected: In one, spots of membrane insertion were visible transiently for an average duration of 340 ms, whereas another class of event showed persistence of about 31 s. Receptors in both categories subsequently appeared to disperse laterally within the membrane with a rate of 3 x 10 –10 square centimeters per second. The β 2 AR stimulates adenylyl cyclase, thus causing accumulation of the second messenger cyclic AMP (adenosine 3′,5′-monophosphate) and activation of protein kinase A (PKA). In cells previously stimulated with isoproterenol for 10 min, inhibition of PKA or removal of the isoproterenol signal rapidly (within the next minute) increased the frequency of the transient insertion events. The more stable insertion events were not affected by inhibition of PKA, but their frequency decreased if the receptor agonist was removed. The functional consequences of these distinct modes of insertion are not yet understood, but the authors propose that they are important for homeostatic regulation of signaling by β 2 ARs and emphasize that trafficking events that modulate receptor abundance may be subject to complex regulation like that controlling neurotransmitter release. G. A. Yudowski, M. A. Puthenveedu, M. von Zastrow, Distinct modes of regulated receptor insertion to the somatodendritic plasma membrane. Nat. Neurosci. 9 , 622-627 (2006). [Online Journal]