Optimizing transmembrane domain helicity accelerates insulin receptor internalization and lateral mobility.
Transmembrane (TM) domains of integral membrane proteins are generally thought to be helical. However, a Gly-Pro sequence within the TM domain of the insulin receptor is predicted to act as a helix breaker. CD analyses of model TM peptides in a lipid-like environment show that substitution of Gly and Pro by Ala enhances helicity. On this basis, Gly933 and Pro934 within the TM domain of the intact human insulin receptor were mutated to Ala (G-->A, P-->A, GP-->AA) to assess effects of altered helicity on receptor functions. Mutated and wild-type receptors, expressed stably in cultured CHO cells at equivalent levels, were properly assembled, biosynthetically processed, and exhibited similar affinities for insulin. Receptor autophosphorylation and substrate kinase activity in intact cells and soluble receptor preparations were indistinguishable. In contrast, insulin-stimulated receptor internalization was accelerated 2-fold for the GP-->AA mutant, compared to a wild-type control or the G-->A and P-->A mutants. Insulin degradation, which occurs during receptor endocytosis and recycling, was similarly elevated in cells transfected with GP-->AA mutant receptors. Fluorescence photobleaching recovery measurements showed that the lateral mobility of GP-->AA mutant receptors was also increased 2- to 3-fold. These results suggest that lateral mobility directly influences rates of insulin-mediated receptor endocytosis and that rates of endocytosis and lateral mobility are retarded by a kinked TM domain in the wild-type receptor. Invariance of Gly-Pro within insulin receptor TM domain sequences suggests a physiologic advantage for submaximal rates of receptor internalization.
Joslin Diabetes Center, Boston, MA 02215.
E
E
Goncalves
K
K
Yamada
H S
HS
Thatte
J M
JM
Backer
D E
DE
Golan
C R
CR
Kahn
S E
SE
Shoelson
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D006224:Cricetinae; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000378:metabolism*; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
D007328:Insulin; D010455:Peptides; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin