Effect Of Caveolae On The Distribution Of CLR In VSMC Membrane

AIM It have been confirmed that many molecules, kinase and most receptors areclustered in the caveolae, and calcitonin receptor-like receptor (CLR), the maincomponent of calcitonin gene-related peptide (CGRP) receptor, which amount of CLRin cell membrane decide the biological effect of CGRP on the cell. Our aim is todetermine whether the caveolae can affect the distribution of CLR in the vascularsmooth muscle cells’ membrane induced by CGRP and whether has a interactionbetween caveolin-1and CLR.METHODS Vascular smooth muscle cells line of mouse source A10were grown at37°C and5%CO2in a humidified incubator in DMEM supplemented with10%fetalbovine serum,0.1%FBS for24h cells’ synchronization. Semi-quantitive RT-PCR wasused to detect the mRNA level of CLR, western blot was used to detect the proteinexpression of CLR. The colocalized of CLR and caveolin-1were detected byimmunofluorescence and confocal microscopy. The interaction between caveolin-1andCLR was determined by immunoprecipitation and Western blotting.RESULTS the distribution of CLR protein in the VSMC membrane reduced byCGRP displayed a time-dependent manner, and the expression of CLR protein levels isheavy decreased at the stimulated fourth hour, and then change little as time extension.CGRP8-37, a CGRP receptor antagonist, can block the CLR decreased effect inducedby CGRP. In addition, the β-cyclodextrin (β-CD), an agent of destroyed caveolae, hadthe same effect as CGRP8-37. It is noticeable that the total levels of CLR protein andmRNA in the β-CD treatment groups were not significant difference.Co-immunoprecipitation experiments detected that CLR had the interaction withcaveolin-1, compared CGRP and0.1%FBS control group, immune complexes of CGRPgroup was reduced. CLR and caveolin-1were colocalized at the plasma membrane byimmunofluorescence and confocal laser microscopy. CONCLUSION The structural integrity of caveolae plays an important role indistribution of CLR, the collapse of caveolae reduced the membrane distribution ofCLR, and this effect may be related to the membrane distribution of CLR/caveolin-1immune complexes.