RECEPTOR-MEDIATED ENDOCYTOSIS OF UROKINASE-TYPE PLASMINOGEN-ACTIVATORIS REGULATED BY CAMP-DEPENDENT PROTEIN-KINASE

Internalization of the urokinase-type plasminogen activator (uPA) requ ires two receptors, the uPA receptor (uPAR) and the low density lipopr otein receptor-related protein (LRP)/alpha(2)-macroglobulin (alpha(2)M ) receptor, Here, we address whether protein kinases are involved in t he internalization of uPA by human melanoma cells. Initially we found that the internalization of uPA was significantly inhibited by the ser ine/threonine protein kinase inhibitors staurosporine, K-252a and H-89 , but not by the tyrosine kinase inhibitors, genistein and lavendustin A, Internalization of uPA was also inhibited by a pseudosubstrate pep tide for cAMP-dependent protein kinase (PKA), but not by a pseudosubst rate peptide for protein kinase C, We confirmed a requirement for PKA- activity and implicated a specific isoform by using an antisense oligo nucleotide against the regulatory subunit RI alpha of PKA which suppre sses PEA-I activity, Exposure of cells to this oligonucleotide led to a specific, dose-dependent decrease in RI alpha protein and to a signi ficant inhibition in the rate of uPA internalization. We further demon strate that treatment of melanoma cells with either H-89 or PKA RI alp ha antisense oligonucleotides also resulted in a decreased internaliza tion of two other ligands of LRP, activated alpha(2)M and lactoferrin, indicating that PKA. activity is associated with LRP. Finally, we dem onstrate that PKA activity is also required for the internalization of transferrin, but not for the internalization of the epidermal growth factor or adenovirus 2, suggesting that in melanoma cells, PKA activit y is not generally required for clathrin-mediated endocytosis, but is rather associated with specific internalization receptors.