Characterization of the extra-large G protein α-subunit XLαs -: I.: Tissue distribution and subcellular localization

Our group previously described a new type of G protein, the 78-kDa XL alphas (extra large as) (Kehlenbach, R. H., Matthey, J., and Huttner, W. B. (1994) Nature 372, 804-809 and (1995) Nature 375, 253). Upon subcellular fractionation, XLas labeled by ADP-ribosylation with cholera toxin was previously mainly detected in the bottom fractions of a velocity sucrose gradient that contained trans-Golgi network and was differentially distributed to G alphas, which also peaked in the top fractions containing plasma membrane. Here, we investigate, using a new antibody specific for the XL domain, the tissue distribution and subcellular localization of XL alphas and novel splice variants referred to as XLN1. Upon immunoblotting and immunofluorescence analysis of various adult rat tissues, XLas and XLN1 were found to be enriched in neuroendocrine tissues, with a particularly high level of expression in the pituitary, By both immunofluorescence and immunogold electron microscopy, endogenous as well as transfected XL alphas and XLN1 were found to be predominantly associated with the plasma membrane, with only little immunoreactivity on internal, perinuclear membranes. Upon subcellular fractionation, immunoreactive XL alphas behaved similarly to G alphas but was differentially distributed to ADP-ribosylated XL alphas. Moreover, the bottom fractions of the velocity sucrose gradient were found to contain not only trans-Golgi network membranes but also certain subdomains of the plasma membrane, which reconciles the present with the previous observations. To further investigate the molecular basis of the association of XL alphas with the plasma membrane, chimeric proteins consisting of the YL domain or portions thereof fused to green fluorescent protein were analyzed by fluorescence and subcellular fractionation, In both neuroendocrine and non-neuroendocrine cells, a fusion protein containing the entire XL domain, in contrast to one containing only the proline-rich and cysteine-rich regions, was exclusively localized at the plasma membrane. We conclude that the physiological role of XL alphas is at the plasma membrane, where it presumably is involved in signal transduction processes characteristic of neuroendocrine cells.