Characterization of the extra-large G protein alpha-subunit XL alpha s - I. Tissue distribution and subcellular localization

Our group previously described a new type of G protein, the 78-kDa XL alpha s (extra large as) (Kehlenbach, R. H., Matthey, J., and Huttner, W. B. (199 4) Nature 372, 804-809 and (1995) Nature 375, 253). Upon subcellular fracti onation, XLas labeled by ADP-ribosylation with cholera toxin was previously mainly detected in the bottom fractions of a velocity sucrose gradient tha t contained trans-Golgi network and was differentially distributed to G alp has, which also peaked in the top fractions containing plasma membrane. Her e, we investigate, using a new antibody specific for the XL domain, the tis sue distribution and subcellular localization of XL alphas and novel splice variants referred to as XLN1. Upon immunoblotting and immunofluorescence a nalysis of various adult rat tissues, XLas and XLN1 were found to be enrich ed in neuroendocrine tissues, with a particularly high level of expression in the pituitary, By both immunofluorescence and immunogold electron micros copy, endogenous as well as transfected XL alphas and XLN1 were found to be predominantly associated with the plasma membrane, with only little immuno reactivity on internal, perinuclear membranes. Upon subcellular fractionati on, immunoreactive XL alphas behaved similarly to G alphas but was differen tially distributed to ADP-ribosylated XL alphas. Moreover, the bottom fract ions 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 in vestigate the molecular basis of the association of XL alphas with the plas ma membrane, chimeric proteins consisting of the YL domain or portions ther eof fused to green fluorescent protein were analyzed by fluorescence and su bcellular fractionation, In both neuroendocrine and non-neuroendocrine cell s, a fusion protein containing the entire XL domain, in contrast to one con taining only the proline-rich and cysteine-rich regions, was exclusively lo calized at the plasma membrane. We conclude that the physiological role of XL alphas is at the plasma membrane, where it presumably is involved in sig nal transduction processes characteristic of neuroendocrine cells.