Phosphotyrosine-containing proteins are minor components of normal cells which appear to be associated primarily with the regulation of cellular metabolism and growth. The insulin receptor is a tyrosine-specific protein kinase, and one of the earliest detectable responses to insulin binding is activation of this kinase and autophosphorylation of its beta-subunit. Tyrosine autophosphorylation activates the phosphotransferase in the beta-subunit and increases its reactivity toward tyrosine phosphorylation of other substrates. When incubated in vitro with [gamma-32P]ATP and insulin, the purified insulin receptor phosphorylates various proteins on their tyrosine residues. However, so far no proteins other than the insulin receptor have been identified as undergoing tyrosine phosphorylation in response to insulin in an intact cell. Here, using anti-phosphotyrosine antibodies, we have identified a novel phosphotyrosine-containing protein of relative molecular mass (Mr) 185,000 (pp185) which appears during the initial response of hepatoma cells to insulin binding. In contrast to the insulin receptor, pp185 does not adhere to wheat-germ agglutininagarose or bind to anti-insulin receptor antibodies. Phosphorylation of pp185 is maximal within seconds after exposure of the cells to insulin and exhibits a dose-response curve similar to that of receptor autophosphorylation, suggesting that this protein represents the endogenous substrate for the insulin receptor kinase.

Amino Acids↗

PubMedDate not supplied

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.

Animals↗

PubMedDate not supplied

A case study in planning for public health education: the organ and tissue donation experience.

The chasm between the supply and demand of donated organs and tissues continues to grow despite widespread public awareness of transplantation and numerous efforts to educate the public about organ donation. It is fast becoming a significant public health problem in this country. The need for more effective public education is well documented in the literature on transplantation and is a primary objective of organizations in the transplant field. In response to this need, the Division of Organ Transplantation in the Health Resources and Services Administration of the Public Health Service initiated a project to examine the nature and scope of donation education initiatives throughout the country, to identify shortcomings, and to suggest ways the Federal Government could contribute to the effectiveness of public education in organ and tissue donation. The project resulted in the development of a protocol that also is applicable to other health education programs. Its major steps consisted of assessing the status of donation-related public education in the United States, identifying existing needs in donation education by applying principles learned from other public health education programs, and identifying roles that could be assumed to help strengthen the American public's commitment to organ and tissue donation. These roles, which could be adopted by an transplant-related organization, were as broker of knowledge, producer of educational strategies, energizer through communications research, and catalyst by bringing together other groups. This approach to needs assessment and planning may provide useful insights both for those concerned with transplants and for professionals conducting education campaigns related to other public health issues.

Health Education↗