The role of receptor kinase activity and the NPEY960 motif in insulin-accelerated receptor-mediated insulin internalization.
This study used biochemical and quantitative ultrastructural approaches to examine the roles that insulin receptor beta subunit kinase activity, the NPEY motif in the juxtamembrane region, and tyrosine phosphorylation within that domain plays in insulin-accelerated receptor-mediated insulin internalization in CHO cells. Internalization of insulin in cells that expressed kinase-deficient receptors (CHOA1018) or receptors lacking the NPEY Ala954-Asp965 domain (CHO delta 960) was reduced by 80% compared to cells expressing wild-type human insulin receptors (CHOHIRc). Ultrastructural analysis revealed that the decreased internalization in CHOA1018 cells was due to the reduced ability of the kinase deficient receptor to migrate from the microvilli of cultured cells and aggregate on the cell surface. Deletion of the NPEY motif in the juxtamembrane region of the beta subunit severely reduced receptor migration, interfered with the normal aggregation of receptors on the cell surface, and virtually eliminated accumulation of the occupied receptors in the coated invaginations. Replacement of Tyr960 in the NPEY domain/with phenylalanine (CHOF960) had no significant effect on insulin internalization, receptor mobility, aggregation or accumulation in coated invaginations. In contrast, replacement of Tyr960 with alanine (CHOA960) decreased insulin internalization, slowed migration, receptor aggregation and accumulation in coated invaginations. These studies document that kinase activity is required, but not sufficient, for receptor movement from the microvilli and aggregation of occupied receptors on the non-villous surface. An intact NPEY motif or surrounding amino acids, but not the phosphorylation of Tyr960 plays a role in receptor mobility and aggregation and is essential for the accumulation of insulin receptors in coated invaginations.
Smith
R M
RM
Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia 19104, USA.
Zhang
S
S
White
M F
MF
Jarett
L
L
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004705:Endocytosis; D006046:Gold; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection
D007328:Insulin; D006046:Gold; D011972:Receptor, Insulin; D017346:Protein Serine-Threonine Kinases; C081827:insulin receptor serine kinase