12.4 Receptor Tyrosine Kinases in Chapter 12 Biochemical Signaling

12.4 Receptor Tyrosine Kinases

The receptor tyrosine kinases (RTKs), a family of plasma membrane receptors with protein kinase activity, transduce extracellular signals by a mechanism fundamentally different from that of GPCRs. RTKs have a ligand-binding domain on the extracellular face of the plasma membrane and an enzyme active site on the cytoplasmic face, connected by a single transmembrane segment, as seen for insulin in Figure 12-21a. The cytoplasmic domain is a Tyr kinase, a protein kinase that phosphorylates specific Tyr residues in target proteins. The receptors for insulin and epidermal growth factor are prototypes for the 58 RTKs in humans (see Fig. 12-26).

A four-part figure shows how the insulin-receptor tyrosine kinase is activated by autophosphorylation by showing the positioning of the inactive receptor with respect to the cell membrane in part a, an illustration showing the locations of bound insulin and phosphate with respect to the two Greek letter alpha subunits and the extracellular portions of two Greek letter beta subunits in part b, a close-up of the inactivate tyrosine kinase domain in part c, and a close-up of the active tyrosine kinase domain in part d. — FIGURE 12-21 Activation of the insulin-receptor tyrosine kinase by autophosphorylation. (a) A structural model assembled from crystal structures of the extracellular and tyrosine kinase domains and NMR solution structure of the transmembrane domain. The insulin-binding region of the insulin receptor lies outside the cell and comprises two intertwined α subunits. (b) The binding of a single molecule of insulin is communicated through the single transmembrane helix of each β subunit to the paired Tyr kinase domains inside the cell, moving them toward each other and activating them to phosphorylate each other on three Tyr residues. (c) In the inactive form of the Tyr kinase domain, the activation loop (backbone shown in teal) sits in the active site, and none of the critical Tyr residues (stick structures) are phosphorylated. This conformation is stabilized by hydrogen bonding between ${Tyr}^{1162}$ $Tyr Superscript 1162$ and ${Asp}^{1132}$ $Asp Superscript 1132$ . **(d)** Activation of the Tyr kinase allows each β subunit to phosphorylate three Tyr residues ( ${Tyr}^{1158}$ $Tyr Superscript 1158$ , ${Tyr}^{1162}$ $Tyr Superscript 1162$ , ${Tyr}^{1163}$ $Tyr Superscript 1163$ ) on the other β subunit. (Phosphoryl groups are depicted in red and orange.) The introduction of three highly charged –Tyr residues forces a 30 Å change in the position of the activation loop, away from the substrate-binding site, which thus becomes available to bind and phosphorylate a target protein. [(a) Data and information from T. Gutmann et al., *J. Cell Biol.* 21:1643, 2018, Fig. 1. Data from (c) PDB ID 1IRK, S. R. Hubbard et al., *Nature* 372:746, 1994; (d) PDB ID 1IR3, S. R. Hubbard, EMBO J. 16:5572, 1997.]

FIGURE 12-21 Activation of the insulin-receptor tyrosine kinase by autophosphorylation. (a) A structural model assembled from crystal structures of the extracellular and tyrosine kinase domains and NMR solution structure of the transmembrane domain. The insulin-binding region of the insulin receptor lies outside the cell and comprises two intertwined α subunits. (b) The binding of a single molecule of insulin is communicated through the single transmembrane helix of each β subunit to the paired Tyr kinase domains inside the cell, moving them toward each other and activating them to phosphorylate each other on three Tyr residues. (c) In the inactive form of the Tyr kinase domain, the activation loop (backbone shown in teal) sits in the active site, and none of the critical Tyr residues (stick structures) are phosphorylated. This conformation is stabilized by hydrogen bonding between ${Tyr}^{1162}$ $Tyr Superscript 1162$ and ${Asp}^{1132}$ $Asp Superscript 1132$ . **(d)** Activation of the Tyr kinase allows each β subunit to phosphorylate three Tyr residues ( ${Tyr}^{1158}$ $Tyr Superscript 1158$ , ${Tyr}^{1162}$ $Tyr Superscript 1162$ , ${Tyr}^{1163}$ $Tyr Superscript 1163$ ) on the other β subunit. (Phosphoryl groups are depicted in red and orange.) The introduction of three highly charged –Tyr residues forces a 30 Å change in the position of the activation loop, away from the substrate-binding site, which thus becomes available to bind and phosphorylate a target protein. [(a) Data and information from T. Gutmann et al., *J. Cell Biol.* 21:1643, 2018, Fig. 1. Data from (c) PDB ID 1IRK, S. R. Hubbard et al., *Nature* 372:746, 1994; (d) PDB ID 1IR3, S. R. Hubbard, EMBO J. 16:5572, 1997.]

Part a shows a horizontal piece of plasma membrane with two long light blue vertical pieces that run across the membrane, with helices in the parts that span the membrane and strands above and below. These pieces expand into rounded ends beneath the membrane. Above the membrane, the light blue pieces join with large light red pieces that run up toward the center and then partially down the other side. The two pieces together form a circular region with an opening and with thicker pieces extending down to the lower left and right to meet the light blue pieces. There are helices and beta strands in the rounded light blue pieces and light red pieces. Part b shows a horizontal piece of plasma membrane with two long light blue vertical pieces that run across the membrane to end in rounded structures. The two strands cross just before they form rounded structures. Each light blue sphere has a rounded rectangular cutout on the upper side facing away from the center. The left-hand light blue circle contains three circles with green borders labeled P. Short lines radiate from all sides of this light blue circle. Above the membrane, the blue pieces widen into light red pieces. The right-hand light red piece curves to the left, then runs up to a rounded piece from which a piece curves to the right and then back behind, where a red sphere labeled insulin is between the front and back pieces. The left-hand light red piece curves to the right, then up, then to the left before curving back across the front. Part c is a close-up of one of the red spheres from part a. It is labeled inactive (unphosphorylated) tyrosine kinase domain. The surface contour of part of a protein is shown. A greenish-blue strand curves from the center top to the left, down the left side, and then up and then down as it runs to the right. Text pointing to this strand reads, activation loop blocks substrate-binding site. Between the top and bottom halves of the greenish-blue strand, there is a stick structure labeled A s p superscript 1132. Extending from below the part of the greenish-blue strand that runs down along the left side, there is a T y r superscript 1158 near the top, then a T y r superscript 1162, then a T y r superscript 1163. Part d shows a close-up from part b. It is similar to the structure in part c except that the greenish-blue strand now runs from the center to curve along the right side and then almost horizontally back across the center, where it ends just before the location of a target protein. A s p superscript 1132 is shown between the top and bottom horizontal pieces of the greenish-blue strand. T y r superscript 1158 is at the point where the strand bends down on the right side, T y r superscript 1162 is at the point where the strand bends left at the lower right, and T y r superscript 1163 is above the strand just to the left of the point where the strand bends left at the lower right. The greenish-blue strand is accompanied by text reading, triply phosphorylated activation loop moves dramatically, making room for the target protein.

Stimulation of the Insulin Receptor Initiates a Cascade of Protein Phosphorylation Reactions

Insulin regulates both metabolic enzymes and gene expression. It initiates a signal that travels divergent pathways from the plasma membrane receptor to insulin-sensitive enzymes in the cytosol, and to enzymes that act in the nucleus by stimulating the transcription of specific genes. The active insulin receptor protein (INSR) consists of two identical α subunits protruding from the outer face of the plasma membrane and two transmembrane β subunits with their carboxyl termini protruding into the cytosol — a dimer of αβ monomers (Fig. 12-21). The α subunits contain the insulin-binding domain, and the intracellular domains of the β subunits contain the protein kinase activity that transfers a phosphoryl group from ATP to the hydroxyl group of Tyr residues in specific target proteins.

Signaling through INSR begins with the binding of one insulin molecule between the two subunits of the receptor on the extracellular side, causing movement of the Tyr kinase domains together and stimulating their activity. Each β subunit phosphorylates three essential Tyr residues near the carboxyl terminus of the other β subunit. This autophosphorylation opens the active site so that the enzyme can phosphorylate Tyr residues of other target proteins. The mechanism of activation of the INSR protein kinase is similar to that described for PKA and PKC: a region of the cytoplasmic domain called the activation loop that usually occludes the active site moves out of the active site after being phosphorylated, opening the site for the binding of target proteins (Fig. 12-21c, d).

When INSR is autophosphorylated (Fig. 12-22, step ) and becomes an active Tyr kinase, one of its targets is the protein insulin receptor substrate 1 (IRS1; step ). Once phosphorylated on several of its Tyr residues, IRS1 becomes the point of nucleation for a complex of proteins (step ) that carry the message from the insulin receptor to end targets in the cytosol and nucleus, through a long series of intermediate proteins. First, a –Tyr residue of IRS1 binds to the SH2 domain of the protein Grb2. Many signaling proteins contain SH2 (Src homology 2) domains, all of which bind –Tyr residues in a protein partner. Grb2 (growth factor receptor-bound protein 2) is an adaptor protein, with no intrinsic enzymatic activity. Its function is to bring together two proteins (in this case, IRS1 and the protein Sos) that must interact to enable signal transduction. In addition to its SH2 (–Tyr-binding) domain, Grb2 contains a second protein-binding domain, SH3, that binds to a proline-rich region of Sos (son of sevenless), recruiting Sos to the growing receptor complex. When bound to Grb2, Sos acts as a guanosine nucleotide–exchange factor (GEF), catalyzing the replacement of bound GDP with GTP on the small G protein Ras.

A figure shows how a M A P kinase cascade is involved in the regulation of gene expression by insulin. — FIGURE 12-22 Regulation of gene expression by insulin through a MAP kinase cascade. The signaling pathway by which insulin regulates the expression of specific genes consists of a cascade of protein kinases, each of which activates the next. The insulin receptor is a Tyr-specific kinase; the other kinases (all shown in blue) phosphorylate Ser or Thr residues. MEK is a dual-specificity kinase that phosphorylates both a Thr residue and a Tyr residue in ERK (extracellular regulated kinase). MEK is mitogen-activated, ERK-activating kinase; SRF is serum response factor.

The upper right cell is shown with the cytosol labeled and the upper right side of the nucleus visible at the lower left. Text above a receptor in the membrane reads, insulin receptor binds insulin and undergoes autophosphorylation on its carboxyl-terminal T y r residues. The receptor has two light red strands that run through the plasma membrane and cross beneath it, where each forms a light blue sphere with a rounded rectangular cutout in the part facing outward. There are three green circles labeled P attached to each light blue sphere. Above the membrane, the thin vertical strands widen. The right-hand strand is labeled Greek letter alpha and curves around to the back. The left-hand strand is labeled Greek letter beta and curves around toward the front, with a red sphere labeled insulin between the back and front pieces at the top. Text reads, insulin receptor phosphorylates I R S 1 on its T y r residues. An orange oval labeled I R S 1 has an arrow pointing up to the circles labeled P bound to the blue spheres at the bottom of the receptor and a second arrow points away to a similar orange circle bonded to three green circles labeled P. Text reads, S H 2 domain of G r b 2 binds to P bond T y r of I R S 1. S o s binds to G r b 2, then to R a s, causing G D P release and G T P binding to R a s. The right-hand P bonded to the orange oval labeled I R S 1 fits into the left side of an irregular gray rectangle labeled G r b 2. The right side of G r b 2 fits into the left side of S o s, which resembles a gray rectangle that is wider at its upper and lower left sides and rounded on the right side where it fits against a green circle labeled R a s. Below R a s, there is a light blue structure labeled R a f-1 that resembles an inverted, blunt pyramid that is rounded above to fit against R a s. R a s has a green thread extending into the plasma membrane to its right. G T P has an arrow pointing to R a s, from which an arrow points to G D P. Accompanying text reads, activated R a s binds and activates R a f-1. A diamond-shaped light blue structure labeled M E K has a curved arrow that runs to R a f-1 and then to a similar diamond-shaped light blue structure with two green circles labeled P attached to its right side. Accompanying text reads, R a f-1 phosphorylates M E K on two S e r residues, activating it. M E K phosphorylates E R K on a T h r and a T y r residue, activating it. A light blue oval labeled E R K has an arrow that curves past M E K with two attached Ps to a light blue oval labeled E R K with two green circles labeled P attached to its right side. Accompanying text reads, E R K moves into the nucleus and phosphorylates nuclear transcription factors such as E l k 1, activating them. An arrow points from E R K with two P to a similar molecule inside the nucleus. A double stranded piece of D N A is shown below. Two pieces that resemble curved orange rectangles that bend down, then up, then down, are labeled S R F and E l k 1. Double-headed arrows run from each of these past E R K with 2 P to similar structures joined together, with S R F on the left and E l k 1 on the right with its right side bonded to a green circle labeled P, on top of the double helix of D N A. A vertical arrow points down from D N A and then to the right, where a green arrow of m R N A is shown with a curved arrow pointing to protein. A white circle is shown below. Accompanying text reads, phosphorylated E L k 1 joins S R F to stimulate the transcription and translation of a set of genes needed for cell division.

In its GTP-bound active form, Ras can activate a protein kinase, Raf-1 (Fig. 12-22, step ), the first of three protein kinases — Raf-1, MEK, and ERK — that form a cascade in which each kinase activates the next by phosphorylation (step ). The protein kinases MEK and ERK are activated by phosphorylation of both a Thr residue and a Tyr residue. When activated, ERK mediates some of the biological effects of insulin by entering the nucleus and phosphorylating transcription factors, including Elk1 (step ), that modulate the transcription of about 100 insulin-regulated genes (step ), some of which encode proteins essential for cell division. Thus, insulin acts as a growth factor.

The proteins Raf-1, MEK, and ERK are members of three larger families, for which several nomenclatures are used. ERK is in the MAPK family (mitogen-activated protein kinases; mitogens are extracellular signals that induce mitosis and cell division). Soon after discovery of the first MAPK enzyme, that enzyme was found to be activated by another protein kinase, which was named MAP kinase kinase (MEK belongs to this family), and when a third kinase that activated MAP kinase kinase was discovered, it was given the slightly ludicrous family name MAP kinase kinase kinase (Raf-1 is in this family). Somewhat less cumbersome are the abbreviations for these three families: MAPK, MAPKK, and MAPKKK. Kinases in the MAPK and MAPKKK families are specific for Ser residues or Thr residues, and MAPKKs (here, MEK) phosphorylate both a Ser residue and a Tyr residue in their substrate, a MAPK (here, ERK).

This insulin pathway is but one instance of a more general scheme in which hormone signals, via pathways similar to that shown in Figure 12-22, result in a change in the phosphorylation of target enzymes by protein kinases or phosphoprotein phosphatases. The target of phosphorylation is often another protein kinase, which then phosphorylates a third protein kinase, and so on. The result is a cascade of reactions that amplifies the initial signal by many orders of magnitude (see Fig. 12-7). MAPK cascades (such as the Raf-MEK-ERK sequence in Fig. 12-22) mediate signaling initiated by a variety of growth factors, such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), both of which are receptor tyrosine kinases like the insulin receptor. Another general scheme exemplified by the insulin receptor pathway is the use of nonenzymatic adaptor proteins, often with intrinsically disordered regions, to bring together the components of divergent signaling pathways, to which we now turn.

The Membrane Phospholipid ${PIP}_{3}$ $PIP Subscript 3$ Functions at a Branch in Insulin Signaling

The signaling pathway from insulin branches at IRS1 (step in Fig. 12-22 and Fig. 12-23). Grb2 is not the only protein that associates with phosphorylated IRS1. The enzyme phosphoinositide 3-kinase (PI3K) binds IRS1 through PI3K’s SH2 domain. Thus activated, PI3K converts the membrane lipid phosphatidylinositol 4,5-bisphosphate $({PIP}_{2})$ $left-parenthesis PIP Subscript 2 Baseline right-parenthesis$ to phosphatidylinositol 3,4,5-trisphosphate ( ${PIP}_{3}$ $PIP Subscript 3$ ) by the transfer of a phosphoryl group from ATP (Fig. 12-24). The multiply (negatively) charged head group of ${PIP}_{3}$ $PIP Subscript 3$ , protruding on the cytoplasmic side of the plasma membrane, is the starting point for a second signaling branch involving another cascade of protein kinases. When bound to ${PIP}_{3}$ $PIP Subscript 3$ , protein kinase B (PKB; also called Akt) is phosphorylated and activated by yet another protein kinase, PDK1 (not shown in Fig. 12-23). The activated PKB then phosphorylates Ser or Thr residues in its target proteins, one of which is glycogen synthase kinase 3 (GSK3). In its active, nonphosphorylated form, GSK3 phosphorylates glycogen synthase, inactivating it and thereby contributing to the slowing of glycogen synthesis. (This mechanism is only part of the explanation for the effects of insulin on glycogen metabolism; see Fig. 15-16.) When phosphorylated by PKB, GSK3 is inactivated. By thus preventing inactivation of glycogen synthase in liver and muscle, the cascade of protein phosphorylations initiated by insulin ultimately stimulates glycogen synthesis (Fig. 12-23).

A figure shows how insulin affects glycogen storage and the movement of G L U T 4 to the plasma membrane. — FIGURE 12-23 Insulin action on glycogen synthesis and GLUT4 movement to the plasma membrane. The activation of PI3 kinase (PI3K) by phosphorylated IRS1 initiates (through protein kinase B, PKB) movement of the glucose transporter GLUT4 to the plasma membrane, and the activation of glycogen synthase.

A plasma membrane runs in a wavy vertical line. A white circle is to the left of a text box reading, G S K 3, inactivated by phosphorylation, cannot converted glycogen synthase (G S) to its inactive form by phosphorylation, so G S remains active. A purple oval labeled G S (inactive) below is bonded to a red circle labeled P to its right. An arrow points to it from a purple oval labeled G S (active) below that has short lines radiating in all directions. An arrow points down from G S active to text reading glycogen above a box reading, synthesis of glycogen from glucose is accelerated. There is a white circle to the left of this text box as well. Next to the upward arrow from G S (active) to G S (inactive), there is a yellow rectangle labeled G S K 3 (active) with short radiating lines from all sides and an arrow that curves past a blue protein labeled P K B that is rounded on the left, bonded to a green circle labeled P on the left, and curved on the right to fit around a green circle labeled P that is attached to a red sphere labeled P I P subscript 3 aligned with the light blue phosphate heads on the inner side of the membrane with two red tails extending into the membrane. Above, an orange oval labeled I R S 1 is bonded to three green circles labeled P. The right-hand P fits into a curve in the upper left side of a roughly rectangular gray protein with curved sides labeled P I 3 K. Accompanying text reads, I R S 1, phosphorylated by the insulin receptor, activates P I 3 K by binding to its S H 2 domain. To the right of P I 3 K, a red sphere labeled P I P subscript 2 is aligned with the light blue phosphate heads on the inner phosphate layer of the membrane and has two red tails extending into the membrane. Below, there is the rounded light blue protein labeled P K B is bonded to a green circle labeled P to its upper left and is curved to fit against a green circle labeled P to its right that is connected to P I P subscript 3 in the membrane. Accompanying text reads, P K B bound to P I P subscript 3 is phosphorylated by P D K 1 (not shown). Thus activated, P K B phosphorylated G S K 3 on a S e r residue, inactivating it. A dashed arrow points down between a green circle labeled R a b on the left and a green circle labeled R A C 1 to the right to an arrow pointing from a vesicle on the left to a region of plasma membrane with two channels on the right. The vesicle has one channel open to the inside and one channel open to the outside. The top channel in the membrane is labeled G L U T 4 and has two curved bars on its sides that each curve away from the center of the channel on the outside of the membrane, opening it to the outside. A light blue hexagon is shown between the two sides where they begin to separate. The bottom channel is open toward the inside. A blue hexagon labeled glucose has an arrow pointing to a glucose hexagon within the cell. Accompanying text reads, P K B, acting through G proteins R A C 1 and R a b, stimulates movement of glucose transporter G L U T 4 from internal vesicles to the plasma membrane, increasing the uptake of glucose.

A figure shows how P I 3 K responds to insulin by producing P I P subscript 3 from P I P subscript 2 and the reverse reaction. — FIGURE 12-24 Regulation of ${PIP}_{3}$ $bold PIP Subscript 3$ formation and breakdown. Phosphoinositide 3-kinase (PI3K) responds to the insulin signal by catalyzing the transfer of a phosphoryl group from ATP to C-3 of the inositol ring of phosphatidylinositol 4,5-bisphosphate $({PIP}_{2})$ $left-parenthesis PIP Subscript 2 Baseline right-parenthesis$ to form phosphatidylinositol 3,4,5-trisphosphate $({PIP}_{3})$ $left-parenthesis PIP Subscript 3 Baseline right-parenthesis$ , which serves as a nucleation point for proteins involved in the MAPK cascade. The enzyme PTEN (phosphatase and *ten*sin homolog) ends the response by catalyzing removal of the same phosphoryl group, which is released as $P_{i}$ $upper P Subscript i$ .

FIGURE 12-24 Regulation of ${PIP}_{3}$ $bold PIP Subscript 3$ formation and breakdown. Phosphoinositide 3-kinase (PI3K) responds to the insulin signal by catalyzing the transfer of a phosphoryl group from ATP to C-3 of the inositol ring of phosphatidylinositol 4,5-bisphosphate $({PIP}_{2})$ $left-parenthesis PIP Subscript 2 Baseline right-parenthesis$ to form phosphatidylinositol 3,4,5-trisphosphate $({PIP}_{3})$ $left-parenthesis PIP Subscript 3 Baseline right-parenthesis$ , which serves as a nucleation point for proteins involved in the MAPK cascade. The enzyme PTEN (phosphatase and *ten*sin homolog) ends the response by catalyzing removal of the same phosphoryl group, which is released as $P_{i}$ $upper P Subscript i$ .

Phosphatidylinositol 4,5-bisphosphate (P I P subscript 2) has a chair conformation of a six-membered ring with the carbons numbered and with the following substituents: C 1 is up-equatorial bonded to O bonded to P double bonded to O to the right, bonded to O minus to the left, and bonded to O above bonded to C H 2 at the left side of a three-carbon chain with the second carbon bonded to H and to O above bonded to C double bonded to O and further bonded to C bonded to R superscript 2 and the third carbon bonded to 2 H and to O above bonded to C double bonded to O and further bonded to C bonded to R superscript 1; C 2 is up-axial bonded to O H; C 3 is up-equatorial bonded to red highlighted O H; C 4 is down-equatorial bonded to O bonded to P bonded to O minus to the right and below and double bonded to O to the left; C 5 is down-equatorial bonded to O bonded to P double bonded to O above and bonded to O minus to the right and below; and C 6 is down-equatorial bonded to O H. An arrow curves right above an irregularly-shaped gray protein labeled P I 3 K with short lines radiating out from all sides. A curved arrow above shows that A T P is added and A D P is removed. The product is phosphatidylinositol 3,4,5-triphosphate (P I P subscript 3). It has a similar structure except that C 3 of the ring is up-axial bonded to O bonded to P bonded to O minus above and to the left and double bonded to O below and C 1 is up-equatorial bonded to O bonded to P double bonded to O to the right, bonded to O minus to the left, and bonded to O above bonded to C H 2 at the right side of a three-carbon chain with the second carbon bonded to H and to O above bonded to C double bonded to O and further bonded to C bonded to R superscript 2 and the third carbon bonded to 2 H and to O above bonded to C double bonded to O and further bonded to C bonded to R superscript 1. A curved arrow labeled P T E N curves back to P I P subscript 2 with an arrow branching away from it to show that red highlighted P subscript i is lost.

In a third signaling branch important in muscle and fat tissue, PKB, acting through two small G proteins (RAC1 and Rab) triggers the clathrin-aided movement of glucose transporters (GLUT4) from intracellular vesicles to the plasma membrane, stimulating glucose uptake from the blood (Fig. 12-23, step ; see also Box 11-1). This increase in glucose uptake triggered by insulin has profound metabolic and medical consequences, as we shall see in Chapter 23.

How fast is the response to insulin? Phosphoproteomics uses high-resolution, high-throughput mass spectrometry to determine, for thousands of proteins in a cell type, which residues in which proteins are phosphorylated in response to a stimulus such as insulin in the living animal (Fig. 12-25). Autophosphorylation of the insulin receptor and IRS1 occurs within a few seconds of insulin addition; protein kinase $C_{β}$ $upper C Subscript beta$ is phosphorylated slightly later (within 15 s), and Sos and Gab are phosphorylated within 0.5 and 1 min. ERK1, the end target in this signaling pathway (Fig. 12-22), is maximally phosphorylated within 3 min. The movement of glucose transporter GLUT4 into the plasma membrane takes about 15 min; and the many changes in gene expression in response to insulin occur over several hours. A recently discovered action of insulin has a different mechanism and a slower time course. In some cases, insulin enters the cell and the nucleus, where, with the help of several other nuclear proteins, it regulates gene expression by binding to promoter regions on the DNA.

A figure shows the time course of phosphorylations triggered by insulin with graphs for each of the following residues: I N S R space Y 1179, I R S 2 space Y 628, P K C Greek letter beta space S 660, S o s 1 space S 1120, G a b 2 space S 2 11, and E R K 1 space T 203/Y 205. — FIGURE 12-25 Time course of phosphorylations triggered by insulin. Mouse liver proteins were examined by mass spectrometry at intervals after injection of insulin to determine quantitatively for each protein which amino acid residues became phosphorylated and when phosphorylation occurred. Each graph represents the phosphorylation of a single residue in the protein, designated by its one-letter abbreviation and position in the primary sequence. Proteins are INSR, insulin receptor; IRS2, insulin receptor substrate 2; PKCβ, protein kinase $C_{β}$ $upper C Subscript beta$ ; Sos and ERK, as in Fig. 12-22. Gab2 is an adaptor protein involved in the MAPK and PI3K signaling pathways. [Data from S. J. Humphrey et al., *Nature Biotechnol.* 33:990, 2015, Fig. 4.]

FIGURE 12-25 Time course of phosphorylations triggered by insulin. Mouse liver proteins were examined by mass spectrometry at intervals after injection of insulin to determine quantitatively for each protein which amino acid residues became phosphorylated and when phosphorylation occurred. Each graph represents the phosphorylation of a single residue in the protein, designated by its one-letter abbreviation and position in the primary sequence. Proteins are INSR, insulin receptor; IRS2, insulin receptor substrate 2; PKCβ, protein kinase $C_{β}$ $upper C Subscript beta$ ; Sos and ERK, as in Fig. 12-22. Gab2 is an adaptor protein involved in the MAPK and PI3K signaling pathways. [Data from S. J. Humphrey et al., *Nature Biotechnol.* 33:990, 2015, Fig. 4.]

Each graph plots fold change on the vertical axis and time in minutes on the horizontal axis. I N S R space Y 1179: The horizontal axis ranges from 0 to 30, labeled in increments of 5, and the vertical axis ranges from 0 to 20, labeled in varying increments. The curve begins at (0, 0.9) and four points are plotted with error bars as follows: (5, 18 plus or minus 1), (10, 16 plus or minus 2), (15, 8 plus or minus 5), (30, 6 plus or minus 3); I R S 2 space Y 628: The horizontal axis ranges from 0 to 30, labeled in increments of 5, and the vertical axis ranges from 0 to 15, labeled in varying increments. The curve begins at (0, 0.9) and four points are plotted with error bars as follows: (5, 12 plus or minus 4), (10, 13 plus or minus 2), (15, 10 plus or minus 4), (30, 7 plus or minus 0.5); P K C Greek letter beta space S 660: The horizontal axis ranges from 0 to 30, labeled in increments of 5, and the vertical axis ranges from 0 to 5, labeled in varying increments. The curve begins at (0, 0.9) and four points are plotted with error bars as follows: (5, 1.9 plus or minus 0.5), (10, 3.5 plus or minus 1.5), (15, 2.5 plus or minus 0.5), (30, 1.5 plus or minus 0.5); S o s 1 space S 1120: The horizontal axis ranges from 0 to 10, labeled in varying increments, and the vertical axis ranges from 0 to 3, labeled in varying increments. The curve begins at (0, 0.9) and seven points are plotted with error bars as follows: (0.5, 1.5 plus or minus 0.1), (1, 1.45 plus or minus 0.2), (2, 1.14 plus or minus 0.4), (3, 1.5 plus or minus 0.3), (4, 1.9 plus or minus 0.6), (6, 1.5 plus or minus 0.4), (10, 1.13 plus or minus 0.4); G a b 2 space S 2 11: The horizontal axis ranges from 0 to 10, labeled in varying increments, and the vertical axis ranges from 0 to 3, labeled in varying increments. The curve begins at (0, 0.9) and five points are plotted with error bars as follows: (0.5, 1.4 plus or minus 0.2), (1, 2.2 plus or minus 0.2), (2, 2.1 plus or minus 0.4), (4, 2.2 plus or minus 0.3), (10, 2.0 plus or minus 0.1); and E R K 1 space T 203/Y 205: The horizontal axis ranges from 0 to 10, labeled in varying increments, and the vertical axis ranges from 0 to 3, labeled in varying increments. The curve begins at (0, 0.9) and seven points are plotted with error bars as follows: (0.5, 2.8 plus or minus 0.2), (1, 2.8 plus or minus 0.3), (2, 2.0 plus or minus 0.3), (3, 2.4 plus or minus 0.3); (4, 2.4 plus or minus 0.4), (6, 1.9 plus or minus 0.1), (10, 2.0 plus or minus 0.6). All data are approximate.

As in all signaling pathways, there is a mechanism for terminating the activity of the PI3K-PKB pathway. A ${PIP}_{3}$ $PIP Subscript 3$ -specific phosphatase (in humans, PTEN) removes the phosphoryl group at the 3 position of ${PIP}_{3}$ $PIP Subscript 3$ to produce ${PIP}_{2}$ $PIP Subscript 2$ (see Fig. 12-24), which no longer serves as a binding site for PKB, and the signaling chain is broken. In several types of cancer, the PTEN gene has often undergone mutation, resulting in a defective regulatory circuit and abnormally high levels of ${PIP}_{3}$ $PIP Subscript 3$ activity. The result is a continuing signal for cell division and thus tumor growth. The unmutated PTEN gene is a tumor suppressor, the subject of Section 12.9.

The insulin receptor is the prototype for several receptor enzymes with a similar structure and RTK activity (Fig. 12-26). The receptors for EGF and PDGF, for example, have structural and sequence similarities to INSR, and both have a protein Tyr kinase activity that phosphorylates IRS1. Many of these receptors dimerize after binding ligand and before autophosphorylation. INSR is the exception, as it is already an ${(α β)}_{2}$ $left-parenthesis alpha beta right-parenthesis Subscript 2$ dimer before insulin binds. (The protomer of the insulin receptor is one αβ unit.) The binding of adaptor proteins such as Grb2 to –Tyr residues is a common mechanism for promoting protein-protein interactions initiated by RTKs, a subject to which we return in Section 12.5.

A figure shows the structures of the following receptor tyrosine kinases: I N S R, V E G F R, P D G F R, E G F R, T r k A, and F G F R. — FIGURE 12-26 Receptor tyrosine kinases. Growth factor receptors that initiate signals through Tyr kinase activity include those for insulin (INSR), vascular endothelial growth factor (VEGFR), platelet-derived growth factor (PDGFR), epidermal growth factor (EGFR), high-affinity nerve growth factor (TrkA), and fibroblast growth factor (FGFR). All these receptors have a Tyr kinase domain (blue) on the cytoplasmic side of the plasma membrane. The extracellular domain is unique to each type of receptor, reflecting the different growth factor specificities. These extracellular domains are typically combinations of structural motifs such as Cys- or Leu-rich segments and segments containing one of several motifs common to immunoglobulins (Ig). Many other Tyr kinase receptors are encoded in the human genome, each with a different extracellular domain and ligand specificity. All of the receptors except INSR are monomeric and their Tyr kinase activity is silent until ligand binding triggers receptor dimerization and Tyr kinase activation. Only INSR is always dimeric, but its Tyr kinase is active only when insulin is bound.

The key shows that a long light blue rectangle with rounded corners is a tyrosine kinase domain, a similar yellow rectangle is a C y s-rich domain, a similar orange rectangle is a L e u-rich domain, a purple rectangle with rounded corners and about one-third of the length is an I g-like domain, and a green elongated circle is an A s p/G l u-rich region. The receptor tyrosine kinases are shown with the ligand-binding domain above a horizontal membrane. The area above the membrane is labeled outside and the area inside the membrane is labeled inside. I N S R: two long light blue rectangles each have short black threads above and below with the threads above running through the membrane to join to the bottoms of the left and right side orange rectangles of a set of four orange rectangles labeled Greek letter beta, each joined with the next by a tiny black thread, and with the outer two positioned slightly lower than the inner two. The inner two orange rectangles each have a short black thread to a yellow rectangle above labeled Greek letter alpha that angles diagonally to the outside to connect by a short black thread to another orange rectangle. V E G F R: a short light blue rectangle has a short black thread below and a short black thread above that joins it to another light blue rectangle that has a black thread that runs through the membrane to join to the bottom short purple rectangle of a vertical chain of seven short purple rectangles with a short black thread extending up from the last one. P D G F R: a short light blue rectangle has a short black thread below and a short black thread above that joins it to another light blue rectangle that has a black thread that runs through the membrane to join to the bottom short purple rectangle of a vertical chain of five short purple rectangles with a short black thread extending up from the last one. E G F R: a light blue rectangle has a short black thread below and a short black thread above that joins it to another light blue rectangle that has a black thread that runs through the membrane to join to a yellow rectangle connected by a black thread to an orange rectangle connected by a black thread to a yellow rectangle connected to another orange rectangle. T r k A: a light blue rectangle has a short black thread below and a short black thread above that joins it to a small purple rectangle above joined by a black thread to another small purple rectangle joined by another black thread to an orange rectangle above with a black thread extending above it. F G F R: a short light blue rectangle has a very short black thread below and a short black thread above that joins it to another short light blue rectangle that has a black thread that runs through the membrane to join to a small purple rectangle above joined by a black thread to a small purple rectangle joined by a black thread to a green circle joined by a black thread to a small purple rectangle with a black thread extending above it.

What spurred the evolution of such complicated regulatory machinery? This system allows one activated receptor to activate several IRS1 molecules, amplifying the insulin signal, and it provides for the integration of signals from different receptors such as EGFR and PDGFR, each of which can phosphorylate IRS1. Furthermore, because IRS1 can activate any of several proteins that contain SH2 domains, a single receptor acting through IRS1 can trigger two or more signaling pathways; insulin affects gene expression through the mitogenic Grb2-Sos-Ras-MAPK pathway and affects glycogen metabolism and glucose transport through the metabolic PI3K-PKB pathway. Finally, there are several closely related IRS proteins (IRS2, IRS3), each with its own characteristic tissue distribution and function, further enriching the signaling possibilities in pathways initiated by RTKs.

Cross Talk among Signaling Systems Is Common and Complex

For simplicity, we have treated individual signaling pathways as separate sequences of events leading to separate metabolic consequences, but there is, in fact, extensive cross talk among signaling systems. The regulatory circuitry that governs metabolism is richly interwoven and multilayered. We have discussed the signaling pathways for insulin and epinephrine separately, but they do not operate independently. Insulin opposes the metabolic effects of epinephrine in most tissues, and activation of the insulin signaling pathway directly reduces signaling through the β-adrenergic signaling system. For example, the INSR kinase directly phosphorylates two Tyr residues in the cytoplasmic tail of a $β_{2}$ $beta 2$ -adrenergic receptor, and PKB, activated by insulin, phosphorylates two Ser residues in the same region (Fig. 12-27). Phosphorylation of these four residues triggers clathrin-aided internalization of the $β_{2}$ $beta 2$ -adrenergic receptor, lowering the cell’s sensitivity to epinephrine.

A figure shows how the insulin receptor and Greek letter beta-adrenergic receptors have interacting effects with activation of I N S R by insulin binding indirectly leading to internalization of the adrenergic receptor. — FIGURE 12-27 Cross talk between the insulin receptor and the β-adrenergic receptor (or other GPCR). When INSR is activated by insulin binding, its Tyr kinase directly phosphorylates the β-adrenergic receptor (right side) on two Tyr residues ( ${Tyr}^{350}$ $Tyr Superscript 350$ and ${Tyr}^{364}$ $Tyr Superscript 364$ ) near its carboxyl terminus, and indirectly causes phosphorylation of two Ser residues in the same region (through activation of protein kinase B (PKB). The effect of these phosphorylations is internalization of the adrenergic receptor, reducing the response to the adrenergic stimulus. Alternatively (left side), INSR-catalyzed phosphorylation of a GPCR (an adrenergic or other receptor) on a carboxyl-terminal Tyr creates the point of nucleation for activating the MAPK cascade (see Fig. 12-22), with Grb2 serving as the adaptor protein. In this case, INSR has used the GPCR to enhance its own signaling.

FIGURE 12-27 Cross talk between the insulin receptor and the β-adrenergic receptor (or other GPCR). When INSR is activated by insulin binding, its Tyr kinase directly phosphorylates the β-adrenergic receptor (right side) on two Tyr residues ( ${Tyr}^{350}$ $Tyr Superscript 350$ and ${Tyr}^{364}$ $Tyr Superscript 364$ ) near its carboxyl terminus, and indirectly causes phosphorylation of two Ser residues in the same region (through activation of protein kinase B (PKB). The effect of these phosphorylations is internalization of the adrenergic receptor, reducing the response to the adrenergic stimulus. Alternatively (left side), INSR-catalyzed phosphorylation of a GPCR (an adrenergic or other receptor) on a carboxyl-terminal Tyr creates the point of nucleation for activating the MAPK cascade (see Fig. 12-22), with Grb2 serving as the adaptor protein. In this case, INSR has used the GPCR to enhance its own signaling.

A horizontal piece of membrane runs across the top of the illustration and the upper left of a nucleus is visible below as a dashed nuclear envelope with cytosol above and nucleus below. An insulin receptor has two light red strands that run through the plasma membrane and cross beneath it, where each forms a light blue sphere with a rounded rectangular cutout in the part facing outward. There are three green circles labeled P attached to each light blue sphere. Above the membrane, the thin vertical strands widen. The right-hand strand is labeled Greek letter alpha and curves around to the back. The left-hand strand is labeled Greek letter beta and curves around toward the front, with a red sphere labeled insulin between the back and front pieces at the top. An arrow points from one of the phosphates to a red circle labeled P that extends down from Y in a red strand extending horizontally from the lower left side of a rectangular structure labeled G P C R that contains a thread that loops up and down. The red circle labeled P is just above a brown structure that is roughly rectangular but curved around the P, narrower on the right, and rounded to fit against the concave left side of an adjacent gray protein labeled G r b 2. G r b 2 has a right side with a concavity into which fits a protrusion from an adjacent gray protein labeled S o s that has a concavity on its right side that fits against a green circle labeled R a s. A light blue protein beneath labeled R a f-1 curves up on either side of R a s. An arrow points from this light blue protein to a light blue diamond labeled M E K from which an arrow points to a blue oval labeled E R K from which an arrow points into the nucleus to text reading, altered gene expression. Another green circle labeled P attached to the bottom of the insulin receptor has an arrow pointing to an orange oval labeled I R S 1 from with an arrow points to a purple protein labeled P K B that is roughly triangular with rounded sides and a concave base. Two arrows point from P K B to two red circles labeled P bonded to two S in a horizontal strand extending from the lower left of a Greek letter beta-adrenergic receptor, which is a light blue rectangle that crosses the membrane and contains a thread that loops up and down. The red thread extending from the lower left of the Greek letter beta-adrenergic receptor has two Ss, both bonded to red circles labeled P as previously described, and then two Ys that are each bonded to red circles labeled P. Arrows point from two green circles labeled P bonded to the blue circles at the bottom of the insulin receptor to the two red circles labeled P bonded to Y. To the right of the Greek letter beta-adrenergic receptor, there is a rounded invagination in the membrane containing a similar Greek letter beta-adrenergic receptor. A dashed line points from the first Greek letter beta-adrenergic receptor to the one in the invagination. Text below reads, G P C R internalization, reduced G P C R responsiveness.

A second type of cross talk between these receptors occurs when –Tyr residues on the $β_{2}$ $beta 2$ -adrenergic receptor, phosphorylated by INSR, serve as nucleation points for SH2 domain–containing proteins such as Grb2 (Fig. 12-27, left side). Activation of the MAPK ERK by insulin is 5- to 10-fold greater in the presence of the $β_{2}$ $beta 2$ -adrenergic receptor, presumably because of this cross talk. Signaling systems that use cAMP and ${Ca}^{2 +}$ $Ca Superscript 2 plus$ also show extensive interaction; each second messenger affects the generation and concentration of the other. Yet another factor that further complicates the signaling picture is that certain metabolic intermediates, such as fatty acids and ceramides, amino acids, and bile acids, can influence insulin signaling. One of the major challenges of systems biology is to sort out the effects of such interactions on the overall metabolic patterns in each tissue — a daunting task.

SUMMARY 12.4 Receptor Tyrosine Kinases

The insulin receptor, INSR, is the prototype of receptor enzymes with Tyr kinase activity. When insulin binds, the receptor Tyr kinase is activated, and phosphorylates Tyr residues on other proteins, such as IRS. Phosphotyrosine residues of IRS1 serve as binding sites for proteins with SH2 domains. These multivalent proteins can serve as adaptors that bring two proteins into proximity. Sos bound to Grb2 activates Ras, which in turn activates a MAPK cascade that ends with the phosphorylation of target proteins in the cytosol and nucleus. The result is specific metabolic changes and altered gene expression.
The enzyme PI3K, activated by interaction with IRS1, phosphorylates the membrane lipid ${PIP}_{2}$ $PIP Subscript 2$ to ${PIP}_{3}$ $PIP Subscript 3$ , which becomes the point of nucleation for proteins in a second and third branch of insulin signaling.
Extensive interconnections among signaling pathways allow integration and fine-tuning of multiple hormonal effects.