©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of the Transmembrane Domain and Flanking Amino Acids in Internalization and Down-regulation of the Insulin Receptor (*)

(Received for publication, October 17, 1994)

Kazunori Yamada Jean-Louis Carpentier (§) Bentley Cheatham Edison Goncalves Steven E. Shoelson C. Ronald Kahn (1)

From the Research Division, Joslin Diabetes Center, the Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02215 Department of Morphology, University of Geneva, 1211 Geneva 4, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have characterized the internalization and down-regulation of the insulin receptor and nine receptors with mutations in the transmembrane (TM) domain and/or flanking charged amino acids to define the role of this domain in receptor cycling. When expressed in Chinese hamster ovary cells, all had normal tetrameric structure and normal insulin-stimulated autophosphorylation/kinase activity. Replacement of the TM domain with that of the platelet-derived growth factor receptor, insertion of 3 amino acids, and substitution of Asp for Val or of Ala for either Gly or Pro had no effect on internalization. Replacement of the TM domain with that of c-neu or conversion of the charged amino acids on the cytoplasmic flank to uncharged amino acids, on the other hand, resulted in a 40-60% decrease in insulin-dependent internalization rate constants. By contrast, substitution of Ala for both Gly and Pro increases lateral diffusion mobility and accelerates internalization rate. These changes in internalization were due to decreased or increased rates of redistribution of receptors from microvilli to the nonvillous cell surface. In all cases, receptor down-regulation and receptor-mediated insulin degradation paralleled the changes in internalization. Thus, the structure of the transmembrane domain of the insulin receptor and flanking amino acids are major determinants of receptor internalization, insulin degradation, and receptor down-regulation.


INTRODUCTION

The insulin receptor is a member of the family of receptor tyrosine kinases and consists of two alpha- and two beta-subunits in an alpha(2)beta(2)-heterotetrameric form(1, 2) . The alpha-subunits are located outside the cell and contain the insulin-binding site. The beta-subunits are transmembrane proteins; each has a 194-amino acid external domain, a single transmembrane (TM) (^1)domain of 23 amino acids, and a large intracellular domain containing the receptor tyrosine kinase(3, 4) . Binding of insulin to the alpha-subunit on the cell-surface receptor results in activation of the beta-subunit kinase, which in turn phosphorylates intracellular substrates such as insulin receptor substrate-1 (IRS-1), initiating the intracellular events that lead to the final biological effects of insulin(5, 6, 7) . At the same time, the insulin-receptor complex undergoes receptor-mediated internalization (8, 9) . Recent studies have demonstrated that insulin-dependent internalization of the receptor requires both an active beta-subunit kinase and an intact intracellular juxtamembrane region(10, 11, 12, 13, 14, 15, 16) . The latter contains sequence motifs, which like those involved in internalization of other receptors, form a tight type I beta-turn exposing at least one aromatic residue(17, 18, 19, 20, 21, 22, 23, 24) .

In this study, we have examined the role of the transmembrane domain and flanking charged amino acids of the insulin receptor in ligand-dependent receptor internalization utilizing a series of nine mutant insulin receptors in which these domains were modified by in vitro mutagenesis. Although these mutants have normal insulin-stimulated receptor kinase activity and an intact juxtamembrane region, using a combination of biochemical and morphological techniques, we find that several of these mutants exhibit decreased insulin-stimulated internalization and one exhibits accelerated internalization. Furthermore, these mutations in the transmembrane domain affect receptor down-regulation and receptor-mediated insulin degradation. Thus, the TM domain and the flanking charged amino acids provide an important determinant of ligand-dependent internalization of the insulin receptor and subsequent receptor down-regulation and ligand degradation.


EXPERIMENTAL PROCEDURES

Materials

I-Insulin was prepared to a specific activity of 3000 Ci/mol at the Peptide Synthesis Core, Joslin Diabetes Center. NaI was purchased from Amersham Corp. Fetal bovine serum and tissue culture medium were from Life Technologies, Inc. Reagents for SDS-polyacrylamide gel electrophoresis were from Bio-Rad. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin was from Worthington. All other reagents including lactoperoxidase, glucose oxidase, soybean trypsin inhibitor, and monensin were purchased from Sigma. Human insulin receptor-specific monoclonal antibody 83-14 directed against the alpha-subunit of the human insulin receptor was kindly supplied by Dr. Kenneth Siddle (Cambridge University, London) and iodinated with IODO-GEN (Pierce) when needed.

In Vitro Mutagenesis and Establishment of CHO Cell Lines Which Overexpress Mutant Insulin Receptors

cDNA sequences encoding human insulin receptors were prepared by oligonucleotide-directed mutagenesis as described previously(18, 25) . Positive candidates were chosen by DNA sequencing the area of interest; selected clones were sequenced over the mutated region plus >50 bases upstream and downstream to confirm the absence of unexpected mutations. Monolayer CHO cells were grown in Ham's F-12 medium containing 10% fetal bovine serum. Cells were cotransfected with a neomycin resistance plasmid and the mutated insulin receptor expression plasmids by calcium phosphate precipitation (25) and selected by growth in the presence of 450 mg/ml Geneticin (Life Technologies, Inc.). Transfected cells were enriched for expression by two or three passages of fluorescence-activated cell sorting using human insulin receptor monclonal antibody 83-14, and clonal cell lines were obtained by limiting dilution. Clones expressing equivalent receptor numbers were identified by insulin binding and Scatchard analyses(25, 26) .

I-Insulin Uptake in CHO Cells Overexpressing Mutant Insulin Receptors

Confluent CHO cells in 24-well plates were incubated with I-insulin (100,000 cpm/well) in internalization medium (serum-free Ham's F-12 medium supplemented with 50 mM Hepes, pH 7.6, and 0.3% bovine serum albumin). After incubation at 37 °C for the indicated times, cells were washed with either PBS at pH 7.4 or PBS at pH 3.5 three times using a 3-min incubation for each wash. Cells were lysed in 0.1 N NaOH, 0.1% SDS, and the radioactivity was counted to determine total cell-associated insulin, i.e. the counts/minutes remaining after the neutral wash, and internalized insulin, i.e. the I-insulin remaining after the acidic wash(13) .

The internalization rate constant (K(e)) was calculated based on the internalization data as described by Lund et al.(27) and Backer et al.(28) . Briefly, the instantaneous velocity of ligand internalization was expressed as dL/dt = K(e)[LR(s)], where L is the amount of internalized ligand and [LR(s)] is the concentration of cell-surface ligand-receptor complex. Integrating both sides of the equation, L = K(e)[LR(s)]dt. Thus, the K(e) is the slope of the line when L is plotted versus [LR(s)] as a function of time. Integrals were approximated by the trapezoidal rule taking an interval of dt = 2 min.

Quantitative Electron Microscopy Autoradiography

Fixed cells from two different experiences were dehydrated, processed for electron microscopy autoradiography, and quantitated as described previously(8, 16, 29) . For each incubation time, three Epon blocks were prepared, and three sections were cut from each block. Thus, for each time point studied for each cell line, 18 separate grids were examined, out of which 1000-2000 grains were analyzed from all cells judged to be morphologically intact. Grains within a distance of 250 nm from the plasma membrane were considered associated with the cell surface, whereas grains overlying the cytoplasm and >250 nm from the plasma membrane were considered internalized. Grains associated with the plasma membrane were divided into the following classes: 1) microvilli, 2) coated pits, 3) nonvillous noncoated pits segments, and 4) uninterpretable. Grains were considered associated with microvilli or coated pits if their center was <250 nm from these surface domains; they were categorized as uninterpretable when the structures underlying the grain could not be unequivocally identified.

Degradation of Insulin by CHO Cells

Confluent CHO cells in 24-well plates were incubated with I-insulin (100,000 cpm/well) for 18 h at 4 °C. Cells were washed twice with chilled PBS and quickly warmed to 37 °C by addition of 0.5 ml of internalization medium to each well. At the indicated times, aliquots of medium were taken and mixed with an equal volume of 15% trichloroacetic acid to give a final trichloroacetic acid concentration of 7.5%. The intact insulin was precipitated by centrifugation in a microcentrifuge, and the radioactivity in the supernatant was counted to determine the amount of degraded insulin. Data are expressed as the ratio of degraded insulin to that initially bound to the cells.

Insulin-induced Down-regulation of Mutant Insulin Receptors

Confluent CHO cells in 24-well plates were incubated with or without 10M insulin at 37 °C for the indicated times in 0.5 ml of internalization medium/well. After incubation, the medium was aspirated, and the cells were washed twice with chilled PBS, pH 3.5, supplemented with 0.3% bovine serum albumin and then twice with PBS, 0.3% bovine serum albumin, pH 7.4, to remove all surface-bound insulin. Each wash included a 3-min incubation. To quantify cell-surface insulin receptors, cells were incubated with either 0.1 ng/ml I-insulin and unlabeled insulin or iodinated monoclonal antibody 83-14 (200,000 cpm/well) in 0.5 ml of internalization medium/well at 4 °C. After 18 h of incubation, cells were washed twice with chilled PBS, pH 7.4, and digested in 0.1 N NaOH, 0.1% SDS, and the associated radioactivity was counted in an automated -counter. In some experiments, cells were preincubated with insulin in the presence of 50 mM monensin, followed by cell-surface receptor quantification.


RESULTS

Insulin Binding and Autophosphorylation of Mutant and Wild-type Receptors

The mutants used in this study include entire replacements of the TM domain by TM domains of the proto-oncoprotein c-neu (TM/c-neu) or the platelet-derived growth factor receptor (TM/PDGFR), an aspartic acid substitution for Val (TM-D/V) analogous to the point mutation in the neu oncoprotein, an insertion of the 3 amino acids Val-Phe-Leu between Leu and Phe, double alanine substitutions for Gly and Pro, single alanine substitutions for either Gly or Pro, and replacements of 3 flanking charged amino acids at the intracellular face of the TM domain (Arg-Lys-Arg) with the uncharged amino acids Ala-Asn-Ala with or without insertion of Arg-Lys-Arg into the exofacial surface of the TM domain (++++exo/cyt and +exo/cyt, respectively). The sequences of all of these mutant TM domains are shown in Fig. 1.


Figure 1: Amino acids sequences of transmembrane domains in normal and mutant insulin receptors. Mutated sequences are underlined.



CHO cells stably transfected with cDNAs for normal and mutant insulin receptors expressed between 2.5 times 10^5 and 3.6 times 10^6 binding sites/cell (Table 1). This is 20-150 times the number of endogenous hamster insulin receptors, estimated to be 1-2 times 10^4/cell. As described previously (25, 30) , each of these receptors was normally processed to give alpha- and beta-subunits, and the affinity for insulin binding in all cases was normal (Table 1). Autophosphorylation of these TM domain mutant insulin receptors in intact cells was also normal as assessed by anti-phosphotyrosine immunoblots of cell extracts after insulin stimulation in vivo (Fig. 2). As a negative control, we also included the Ala receptor mutant, which has an alanine for lysine substitution in the ATP-binding site and is known to be a kinase-defective mutant (19, 31, 32) (data not shown).




Figure 2: Autophosphorylation of TM domain mutant insulin receptors in intact cells. Confluent CHO cells expressing mutant and wild-type insulin receptors were incubated with 10M insulin in Ham's F-12 medium at 37 °C for 5 min. Cells were then solubilized as described under ``Experimental Procedures,'' and insulin receptors were immunoprecipitated with antibody 83-14. Tyrosine phosphorylation of the insulin receptor was detected by immunoblotting with anti-phosphotyrosine antibody. In all cases, the odd-numbered lanes represent basal phosphorylation, and the even-numbered lanes represent insulin-stimulated samples. Representative experiments are show for the wild-type receptor (A, lanes 1 and 2; B, lanes 1 and 2; and C, lanes 1 and 2), +exo/cyt (A, lanes 3 and 4), ++++exo/cyt (A, lanes 5 and 6), TM/c-neu (B, lanes 3 and 4), TM/PDGFR (B, lanes 5 and 6), TM-D/V (B, lanes 7 and 8), TM+3 (B, lanes 9 and 10), TM-AA/GP (C, lanes 3 and 4), TM-A/P (C, lanes 5 and 6), and TM-A/G (C, lanes 7 and 8).



Insulin Uptake in CHO Cells

As with most cell-surface receptors, following insulin binding at physiological temperature (37 °C), some of the occupied insulin receptors are internalized along with the bound insulin. This pool of internalized bound insulin can be distinguished from the cell-surface pool by resistance to elution with an acidic wash as described under ``Experimental Procedures.'' The time course of internalization at 37 °C for all the TM domain mutants, the wild-type receptor, and the Ala mutant is shown in Fig. 3A. As described previously(10, 11, 12, 15, 16) , the kinase-defective Ala mutant showed little or no insulin-stimulated internalization over the time course of study. Among the TM domain mutants studied, TM/c-neu, +exo/cyt and ++++exo/cyt also showed reduced levels of I-insulin uptake when compared with the wild-type receptors. By contrast, internalization of insulin receptors was increased by 10-100% for the TM-AA/GP receptor mutant (Fig. 3A, right panel). Internalization was also studied in the CHO cells expressing normal insulin receptors and selected TM domain mutants using quantitative autoradiography at the electron microscopic level (Fig. 3B). Again, by this method, internalization was impaired for both the TM/c-neu and ++++exo/cyt mutants and increased in the TM-AA/GP mutant (Fig. 3B). Morphological and biochemical analysis also indicated that the inhibition of I-insulin internalization observed in TM/c-neu and ++++exo/cyt CHO cells was not as complete as that observed in cells expressing the Ala insulin receptor mutant (Fig. 3, A and B).



Figure 3: Internalization of I-insulin. CHO cells expressing mutant and wild-type (WT) receptors were incubated with I-insulin (100,000 cpm/well) at 37 °C for the indicated times. Cells were washed with either PBS at pH 7.4 or PBS at pH 3.5, after which the cell-associated radioactivity was determined. In A, the acid-resistant radioactivity was considered as internalized. In B, the percentage of the total number of autoradiographic grains seen associated with the cells at the electron microscopic level that are centered >250 nm from the plasma membrane represents the percentage I-insulin internalized. In these experiments, cells were continuously incubated in the presence of I-insulin for the indicated periods of time at 4 or 37 °C. Results concerning Ala mutant insulin receptor are derived from (16) .



The K(e) values were calculated from the acid wash data as described previously (28) (Fig. 4). The K(e) values for TM domain mutants TM/c-neu, +exo/cyt, and ++++exo/cyt were decreased by 40-60% as compared with that of the wild-type receptor (Fig. 4). The Ala mutant, which has abolished tyrosine kinase activation, showed the lowest level internalization rate (K(e) 15% of the wild-type K(e)). On the other hand, the TM-AA/GP insulin receptor mutant, in which 2 naturally occurring helix-breaking amino acids (Gly and Pro) were replaced with 2 helix-favoring alanines, was found to have an accelerated insulin uptake rate corresponding to 70% above that of the wild-type receptor (Fig. 4, A, rightpanel, opensquares; and B). All other mutant receptors showed a normal insulin uptake rate ( Fig. 3and Fig. 4).



Figure 4: Rate constant for insulin internalization. The K values for each mutant and wild-type (WT) receptor were determined as described under ``Experimental Procedures.'' Data represent the means ± S.E. of three to six separate experiments. LR(s), concentration of cell-surface ligand-receptor complex



Surface Localization of the Insulin Receptor

Two surface steps precede the entry of insulin receptors inside the cells: the first is ligand-specific and consists of the insulin-induced redistribution of the receptor from microvilli to the nonvillous domain of the cell surface; the second is ligand-independent and common to many receptors and consists of the anchoring of receptors to clathrin-coated pits, which represent the internalization gate(16) . To distinguish whether the modified rates of internalization observed with the various insulin receptor TM domain mutants were related to altered surface events, we analyzed morphologically the surface localization and redistribution of these receptors. With respect to the first step, both the TM/c-neu and ++++exo/cyt mutants exhibited defects in internalization and redistributed from microvilli to the nonvillous region of the cell surface in response to I-insulin binding more slowly and to a lesser extent than wild-type receptors, but faster than the Ala mutant receptors (Fig. 5). By contrast, the TM-AA/GP mutant receptor, which exhibited an increased internalization rate, was more rapidly redistributed than the normal human insulin receptor (Fig. 5).


Figure 5: Surface redistribution of I-insulin in CHO cells expressing mutant and wild-type receptors. Results presented are the means ± S.E. of the analysis of three different Epon blocks from two different experiments (n = 6). For each time point and each cell line, 2000 autoradiographic grains were quantitated. Cells were continuously incubated in the presence of I-insulin for the indicated periods of time at 4 or 37 °C. Results concerning the Ala mutant insulin receptor are derived from (16) . WT, wild-type receptor.



With respect to the second step of internalization, the anchoring in clathrin-coated pits, the TM/c-neu and ++++exo/cyt mutants exhibited a reduced association with these surface areas when all surface-associated autoradiographic grains localizing I-insulin were considered (Fig. 6A). However, if we considered only labeled material present on the nonvillous cell surface, these receptor mutants showed the same propensity to associate with clathrin-coated pits (Fig. 6B). Thus, these mutants are able to anchor to clathrin-coated pits, but are not concentrated in these surface invaginations because they do not have access to the surface domain of the cell surface where these structures are located. In the case of the TM-AA/GP receptors, an increased association with clathrin-coated pits was observed whichever mode of calculation was used (Fig. 6, A and B). This suggests that the increased mobility of the TM-AA/GP receptor also increased its capacity to anchor to clathrin-coated pits.


Figure 6: Association of I-insulin present on the total cell surface (A) or on the nonvillous surface (B) with clathrin-coated pits in CHO cells expressing mutant and wild-type receptors. Results presented are the means ± S.E. of the analysis of three different Epon blocks from two different experiments (n = 6). For each time point and each cell line, 2000 autoradiographic grains were quantitated. Cells were continuously incubated in the presence of I-insulin for the indicated periods of time at 4 or 37 °C. Results concerning the Ala mutant insulin receptor are derived from (16) . WT, wild-type receptor.



Insulin Degradation

Fig. 7shows the degradation of I-insulin by CHO cells expressing the four mutant receptors that exhibited decreased or accelerated insulin uptake as well as the wild-type receptor and the Ala mutant. For the wild-type receptor, there was relatively little insulin degradation over the first 10 min of incubation and then an increase in rate such that 24% of the cell-associated insulin was degraded after 30 min. The TM/c-neu, +exo/cyt, and ++++exo/cyt receptors showed a reduced level of insulin degradation (ranging from 12 to 18% at 30 min), whereas the TM-AA/GP mutant had increased insulin degradation compared with the wild-type receptor (36% at 30 min). The kinase-defective Ala mutant showed the lowest level of insulin degradation (11% at 30 min). Thus, the amount of degraded insulin changed in proportion to the level of insulin uptake.


Figure 7: Insulin degradation in CHO cells. CHO cells expressing mutant and wild-type (WT) receptors were incubated with I-insulin at 4 °C for 18 h. Cells were washed with chilled PBS and warmed up to 37 °C in Ham's F-12 medium. After incubation for the indicated times at 37 °C, the amount of degraded insulin was determined by trichloroacetic acid precipitation. Data are expressed as a percentage of initial bound insulin.



Down-regulation of Mutant Insulin Receptors

Preincubation of CHO cells with high concentrations of insulin (10M) causes a loss of cell-surface insulin receptor, i.e. down-regulation in a time-dependent manner. Fig. 8shows a representative time course for down-regulation of I-insulin binding with CHO cells that overexpress wild-type insulin receptors. After 16 h of incubation, the bound/free ratio for I-insulin decreased by 35% as compared with that in cells incubated with buffer but without insulin for 16 h. Each mutant receptor also showed reduced surface binding of insulin after 16 h of preincubation with insulin; however, the degree of down-regulation was only 22, 18, and 15% for +exo/cyt, ++++exo/cyt, and TM/c-neu, respectively, all of which showed reduced internalization. As with other parameters, the TM-AA/GP mutant receptor showed an increased extent of down-regulation, which was 50% greater than that of the wild-type receptor. These changes in binding were due to changes in cell-surface receptor concentration, as demonstrated by Scatchard analysis (data not shown). When assessed by insulin binding, the kinase-defective mutant receptor Ala showed an unexplained, but reproducible, small increase in ligand binding following prolonged insulin pretreatment.


Figure 8: Down-regulation of mutant receptors (preincubation time course). CHO cells were preincubated with or without 10M insulin for the indicated times at 37 °C. Cells were washed twice with PBS at pH 3.5 and twice with PBS at pH 7.4 to remove surface-bound insulin. Cells were then incubated with I-insulin for 18 h at 4 °C. After washing, cells were digested, and the associated radioactivity was counted. Data are expressed as the bound/free ratio of ``down-regulated'' to control. Each point represents the mean ± S.E. of three separate experiments.



Loss of cell-surface receptor was also estimated by changes in binding of labeled anti-receptor antibody (Fig. 9). After preincubation with 10M insulin for 18 h, binding of iodinated monoclonal antibody 83-14 was decreased by 30% for the wild-type receptor. The +exo/cyt, ++++exo/cyt, and TM/c-neu mutations showed reduced antibody binding by 19, 20, and 24%, respectively, whereas the TM-AA/GP mutant had a 41% decrease in antibody binding with insulin preincubation. These data confirm the fact that the loss of insulin binding is due to a loss of immunoreactive receptor protein, rather than some modification of the insulin-binding site. Furthermore, the degree of insulin-induced receptor down-regulation varied in proportion to the change in receptor internalization in each receptor mutant. In contrast to the studies with labeled insulin, however, no change in receptor was found in cells expressing the Ala mutant when assessed by antibody binding.


Figure 9: Down-regulation as measured by anti-receptor antibody binding. CHO cells were preincubated with or without 10M insulin and washed as described in the legend to Fig. 8. Cells were incubated with I-labeled monoclonal antibody 83-14 to the insulin receptor (2 times 10 cpm/well) at 4 °C for 18 h. Cells were washed with PBS and digested, and the associated radioactivity was counted. WT, wild-type receptor.



Effect of Monensin on Insulin-induced Down-regulation of Mutant Receptors

Insulin-induced receptor down-regulation is a reflection not only of receptor endocytosis, but also of synthesis, recycling, and degradation. Monensin, a calcium ionophore that inhibits receptor recycling without affecting internalization(33) , was used to further investigate the relationship between internalization and down-regulation of mutant insulin receptors. In this experiment, preincubation of CHO cells with 10M insulin for 3 h at 37 °C caused receptor down-regulation by 27% for the wild-type receptor and between 6 and 39% for the various mutants (Table 2). When cells were incubated with insulin in the presence of 50 mM monensin for 3 h, in each case, loss of cell-surface receptors became even greater than that without monensin. For example, with monensin, the wild-type receptor was down-regulated by 36%. One can obtain an estimate of the ``recycled fraction'' by calculating the percent increase in down-regulation in the presence versus the absence of monensin, and for the wild-type receptor, this value was 28%. Based on similar calculations, the recycled fraction of internalized receptors was estimated to be between 23 and 36% for all cell lines. Thus, there was no significant difference in recycling of the internalized receptor to the cell surface among mutant and wild-type receptors, suggesting that the differences in receptor down-regulation can be accounted for mainly by the differences in internalization among these receptors.




DISCUSSION

Internalization of membrane receptors may play a critical role in their cellular function, as for example with the receptor for many nutrient-related components such as low density lipoprotein and transferrin, or may play a more regulatory role, as in the case of most hormone receptors. Evidence that the insulin receptor is involved in ligand-stimulated internalization has been present for over 15 years, but only recently have the structural determinants of this process begun to be elucidated. The present results clearly show the importance of the transmembrane domain of the insulin receptor in ligand-dependent receptor internalization as demonstrated by a decreased receptor internalization in the TM/c-neu mutant and an increased internalization in the TM-AA/GP mutant. Our data also indicate that the 3 positively charged amino acids on the cytoplasmic flank of the transmembrane domain are another determinant of receptor internalization since replacement of this sequence with uncharged amino acids (+exo/cyt and ++++exo/cyt mutations) results in impaired internalization. These findings could be confirmed measuring I-insulin internalization both biochemically and morphologically and by performing insulin degradation and insulin receptor down-regulation studies.

These determinants of insulin-dependent receptor internalization are independent of receptor kinase activation or structure of the juxtamembrane region, both of which have been reported to be critical components in receptor internalization(10, 11, 12, 13, 14, 15, 16, 17, 18) , since all mutants examined had normal kinase activation and an intact juxtamembrane domain. Regulation of internalization independent of kinase activation has also been suggested by Trischitta et al.(34) , who showed that insulin receptor antibodies that do not activate receptor tyrosine kinase can also induce receptor internalization, and by Androlewicz et al.(35) , who reported an insulin-resistant melanoma cell line that exhibited decreased insulin-induced receptor internalization despite normal ligand-dependent kinase activation. Along with present results, these observations indicate that the mechanisms by which ligand-dependent internalization can be regulated are complex and that the structure of the TM domain and its flanking charged amino acids are major components in the regulation of this system.

The uptake of insulin-receptor complexes inside target cells is preceded by surface events that can be subdivided in three steps: 1) freeing of the receptor from microvilli, where they preferentially localize in the absence of bound ligand; 2) surface redistribution from microvilli to the nonvillous domain of the cell surface; and 3) anchoring of the receptor in clathrin-coated pits(15, 16) . The first of these events is ligand-specific and depends on receptor kinase activation and autophosphorylation, while the third requires specific signal sequences present in the juxtamembrane domain of the receptor (15, 16, 17, 18) . Neither of these two events seems affected by the mutations used in the present study since all TM domain receptor mutants analyzed have a normally activable kinase, intact autophosphorylation sites, and preserved juxtamembrane domains. Moreover, as demonstrated by the morphological analysis, the three TM domain receptor mutants that exhibit decreased or increased internalization can leave microvilli and show a normal propensity to associate with clathrin-coated pits on the nonvillous domain of the cell surface. This is in contrast to what has been previously observed in the case of kinase-inactive or juxtamembrane mutated receptors(15, 16) . Thus, the altered surface redistribution of these three receptor mutants (TM-AA/GP, ++++exo/cyt, and TM/c-neu) most probably reflects altered surface mobility of these receptors. Indeed, using fluorescence photobleaching, the TM-AA/GP receptors have been shown to exhibit increased lateral mobility within the plane of the plasma membrane(30) . This suggests that the structure of the transmembrane domain may play an important role in the interaction with the phospholipid bilayer of the plasma membrane. It is interesting to note that internalization and intracellular processing of insulin receptors are reduced in type II diabetes and obesity (36, 37, 38, 39, 40) , disorders in which there is a change in lipid composition of the membrane (41, 42, 43) as well as decreased receptor kinase activation.

The exact role of internalized receptors in insulin action remains uncertain(44, 45, 46, 47) . The fact that double alanine substitution for Gly and Pro (TM-AA/GP) exhibited an accelerated ligand-dependent internalization suggests that the native structure of the transmembrane domain does not necessarily provide the best conformation for ligand-dependent internalization. This further suggests that internalization of the insulin receptor may be regulated in some way that provides the best efficiency for transmission of the extracellular signal to intracellular receptor kinase domain.

Another important finding in this study is a tight correlation between insulin-dependent receptor internalization and insulin-induced receptor down-regulation in each mutation. Internalization-defective mutations, which include TM/c-neu, +exo/cyt, and ++++exo/cyt mutants, all exhibited decreased levels of insulin receptor down-regulation and insulin degradation, whereas the TM-AA/GP mutant, which had enhanced receptor internalization, showed accelerated receptor down-regulation and increased insulin degradation. Correlations among internalization, receptor down-regulation, and insulin degradation have also been observed with chimeric receptor constructs and other mutants(48, 49, 50) . Moreover, in studies analogous to the present one, we (15) and others (10, 51) reported similar observations in the case of various mutations of the insulin receptor accompanied by changes in receptor internalization rate and magnitude. Although insulin-induced receptor down-regulation is thought to be a complicated phenomenon that includes not only the endocytosis, but also degradation, recycling, and biosynthesis of the insulin receptors(45, 46) , we conclude that insulin-dependent receptor internalization is the major factor in insulin-induced down-regulation and intracellular insulin degradation.


FOOTNOTES

*
This work was supported by Grant DK 31036 from the National Institutes of Health, by Grant 31.34093.92 from the Swiss National Science Foundation, by the Juvenile Diabetes Foundation, and by the core laboratories funded by Diabetes and Endocrinology Research Center Grant DK 36836. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

()
To whom correspondence should be addressed: Research Div., Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. Tel.: 617-732-2635; Fax: 617-732-2593.

(^1)
The abbreviations used are: TM, transmembrane; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We thank C. Maeder and G. Berthet for skilled technical assistance and Terri-Lyn Bellman and Nanda E. Barker for excellent secretarial assistance.


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