ROLES FOR A CYTOPLASMIC TYROSINE AND TYROSINE KINASE-ACTIVITY IN THE INTERACTIONS OF NEU RECEPTORS WITH COATED PITS

The neu proto-oncogene product, p185(neu) (HER2, c-ErbB-2), encodes a cell-surface tyrosine kinase receptor with high oncogenic potential, w hich correlates with increased tyrosine kinase activity and a rapid re ceptor internalization rate, To investigate the interactions and signa l(s) leading to the endocytosis of Neu receptors, we employed lateral mobility and internalization studies, Fluorescence photobleaching reco very measurements revealed that activation of Neu receptors (induced b y mutation or by agonistic antibodies) markedly reduced their mobile f ractions, To elucidate the signals involved, other mutants, all carryi ng a constitutively dimerizing oncogenic mutation, were analyzed, A ki nase-negative mutant and a mutant lacking all cytoplasmic tyrosine pho sphorylation consensus sequences exhibited high mobile fractions, simi lar to nonactivated Neu, Retention of a single tyrosine autophosphoryl ation site (Tyr-1253) out of the five known such sites was sufficient to immobilize a large fraction of the receptor, For all mutants, inter nalization correlated with receptor immobilization and was blocked by treatments that interfere with coated pit structure, indicating that t he immobilization is due to interactions with coated pits, This was su pported by the coimmunoprecipitation of alpha-adaptin only with the co nstitutively activated Neu mutants, We conclude that activated Neu rec eptors become stably associated with coated pits via plasma membrane a daptor complexes (AP-2), Efficient Neu receptor endocytosis requires a ctivation, a functional kinase domain, and at least one tyrosine autop hosphorylation site.