Role of lysine187 within the second extracellular loop of the type A cholecystokinin receptor in agonist-induced activation. Use of complementary charge-reversal mutagenesis to define a functionally important interdomain interaction

Abstract

Activation of guanine nucleotide-binding protein (G protein)-coupled receptors is believed to involve conformational change that exposes a domain for G protein coupling at the cytosolic surface of the helical confluence, although the mechanisms for achieving this are not well understood. This conformational change can be achieved by docking a diverse variety of agonist ligands, known to occur by interacting with different regions of these receptors. In this study, we focus on the importance of a specific basic residue (Lys187) within the second extracellular loop of the receptor for the peptide hormone, cholecystokinin. Alanine-replacement and charge-reversal mutagenesis of this residue showed that it had no effect on the binding of natural peptide and nonpeptidyl ligands of this receptor but markedly interfered with agonist-stimulated signaling. It was demonstrated that this negative effect on biological activity could be eliminated with the truncation of the first 30 residues of the amino-terminal tail of this receptor. Complementary charge-reversal mutagenesis of each of the five conserved acidic residues within this region of the receptor in the presence of the charge-reversed Lys187 revealed that only the Asp5 mutant fully reversed the negative functional impact of the Lys187 charge reversal. Thus, we have demonstrated that a basic residue within the second extracellular loop of the cholecystokinin receptor interacts with a specific acidic residue within the amino terminus of this receptor. This residue-residue interaction is nicely accommodated within a new molecular model of the agonist-occupied cholecystokinin receptor.

FIGURE 1

Schematic diagram of the type A CCK receptor and the sites of modification in various key receptor constructs used in this project.

FIGURE 2

Effects of mutation of CCK receptor residue Lys¹⁸⁷ on binding of peptide and non-peptidyl ligands. Top left, curves for CCK to compete for binding of the ¹²⁵I-CCK radioligand to membranes from CHO cells expressing the wild type, K187A, and K187D CCK receptor constructs. Top right, curves for non-peptidyl agonist, 1,5-benzodiazepine #1d, to compete for binding of the ¹²⁵I-CCK radioligand to these cell membranes. Bottom left, curves for non-peptidyl antagonist, L-364,718, to compete for binding of the ³H-L-364,718 radioligand to these cell membranes. Bottom right, curves for non-peptidyl antagonist, SR-27897, to compete for binding of the ³H-SR-27897 radioligand to these cell membranes. Data are presented as means ± S.E.M. of data from a minimum of three independent experiments. Table 1 includes K_i and B_max values for these series of studies. Mutation of CCK receptor residue Lys¹⁸⁷ to either a neutral charge (Ala) or a reversed-charge (Asp) residue had no significant effects on ligand binding of either peptidyl agonist CCK or non-peptidyl agonist, 1,5-benzodiazepine #1d or antagonists, L-364,718 and SR-27897.

FIGURE 3

Effects of mutation of the CCK receptor residue Lys¹⁸⁷ on biological activity of peptide and non-peptidyl ligands. Shown are intracellular calcium responses in CHO cells expressing the wild type, K187A, and K187D CCK receptor constructs to increasing concentrations of CCK (left) and non-peptidyl ligand, 1,5-benzodiazepine #1d (right). Data are presented as means ± S.E.M. of data from a minimum of three independent experiments. Basal level of intracellular calcium was similar for all the constructs (157 ± 26 nM), and maximal levels of stimulation by CCK reached 393 ± 33 (WT), 389 ± 47 (K187A) and 277 ± 33 (K187D) nM and by 1,5-benzodiazepine #1d reached 370 ± 29 (WT) and 355 ± 40 (K187A) nM. EC₅₀ and E_max (delta change from basal) values are shown in Table 1. The CCK-stimulated intracellular calcium response was decreased about 4-fold by neutral charge K187A and 233-fold by reversed-charge K187D mutations. The 1,5-benzodiazepine #1d-stimulated calcium response was decreased about 20-fold by neutral charge K187A mutation, while the reversed-charge K187D substitution completely abolished the response.

FIGURE 4

Morphological evidence for normal cell surface expression of the Lys¹⁸⁷ mutant CCK receptor constructs. Shown are representative examples of CHO cells expressing the wild type CCK receptor in the absence (CHO-WT) and presence of competing CCK (CHO-WT + CCK), as well as K187A (CHO-K187A) and K187D (CHO-K187D) CCK receptor mutants, and non-receptor-bearing parental CHO cells labeled with Alexa-CCK. There was no significant surface labeling of either the parental CHO cells or the receptor-bearing CHO-WT cells in the presence of competing non-fluorescent CCK. All images were acquired under similar settings and were representative of at least three independent experiments.

FIGURE 5

Time course of internalization of the Lys¹⁸⁷ mutant CCK receptor constructs in CHO cells. CHO cells stably expressing the wild type (WT), K187A, and K187D CCK receptor constructs were preincubated for 1 h at 4 °C with 50 nM Alexa-CCK, and were subsequently washed and warmed to 37 °C for 0, 2, 5, 10 and 30 min before being fixed. Images are representative of three independent experiments. Mutation of CCK receptor residue Lys¹⁸⁷ to either a neutral charge (Ala) or a reversed-charge (Asp) residue had no significant effects on the extent or time-course of CCK receptor internalization.

FIGURE 6

Effects of truncation and site mutation of the amino terminus of the K187D mutant CCK receptor construct on their peptidyl ligand binding and biological activity. Left, curves for CCK to compete for binding of the ¹²⁵I-CCK radioligand to membranes from CHO cells expressing the truncated WT ((WT)31–429) and K187D ((K187D)31–429) CCK receptor constructs (top) and membranes from COS cells expressing the site mutants, D5K and D5K(K187D) (bottom). As controls, data from WT and K187D are also shown. K_i and B_max values are shown in Table 1. Right, curves of intracellular calcium responses in these cells to increasing concentrations of CCK. Data are presented as means ± S.E.M. of data from a minimum of three independent experiments. Basal levels of intracellular calcium in CHO cells were similar for all the constructs (147 ± 28 nM), and maximal levels achieved after stimulation with CCK reached 380 ± 40 (WT), 375 ± 36 ((WT)31–429) and 387 ± 49 ((K187D)31–429) nM. There were no significant differences in basal and maximal intracellular calcium levels for any of the CCK receptor mutants expressed in COS cells. In those cells, basal levels of intracellular calcium were 166 ± 34 nM, and maximal levels achieved after stimulation with CCK were 268 ± 43 nM. EC₅₀ and E_max (delta change from basal) values are shown in Table 1. These data suggest that neither truncation nor site mutation of the amino terminus of the K187D mutant CCK receptor had any effect on peptidyl ligand binding, while both completely restored the intracellular calcium responses to CCK.

FIGURE 7

Effects of truncation and site mutation of the amino terminus of the Lys¹⁸⁷ mutant CCK receptor constructs on their non-peptidyl ligand binding and biological activity. Shown in left panels are curves for 1,5-benzodiazepine #1d to compete for binding of the ¹²⁵I-CCK radioligand to membranes from CHO cells expressing the truncated WT ((WT)31–429) and K187D ((K187D)31–429) CCK receptor constructs (top) and membranes from COS cells expressing the site mutants, D5K and D5K(K187D) (bottom). As controls, data from WT and K187D are also shown. K_i and B_max values are shown in Table 1. Shown in right panels are curves of intracellular calcium responses in these cells to increasing concentrations of 1,5-benzodiazepine #1d. Data are presented as means ± S.E.M. of data from a minimum of three independent experiments. Basal levels of intracellular calcium in CHO cells were similar for all the constructs (143 ± 21 nM), and maximal levels achieved after stimulation with 1,5-benzodiazepine #1d were 363 ± 49 (WT), 333 ± 29 ((WT)31–429) and 320 ± 33 ((K187D)31–429) nM. No significant differences in basal or maximal intracellular calcium levels were observed for any of the CCK receptor mutants expressed in COS cells. Basal levels of intracellular calcium were 160 ± 40 nM, and maximal levels achieved after stimulation with 1,5-benzodiazepine #1d were 262 ± 47 nM. These data demonstrate that amino-terminal truncation and site mutation of the K187D mutant CCK receptor are both able to restore intracellular calcium responses to 1,5-benzodiazepine #1d. Both constructs bound normally.

FIGURE 8

Molecular model of CCK bound to the type A CCK receptor. Shown are lateral (left) and top (right) views of the lowest energy molecular model of CCK bound to the CCK receptor. The receptor amino-terminal domain is illustrated in brown, while the rest of the receptor is green. The peptide ligand, CCK-8, is shown as a stick model with transparent surface skin in white. Key residues of interest have been highlighted. These include receptor residues Asp⁵ and Lys¹⁸⁷ that are involved in charge-pairing, Cys pairs that are involved in disulfide bonds (Cys¹⁸-Cys²⁹ and Cys¹¹⁴-Cys¹⁹⁶), and three sites of N-linked glycosylation (Asn¹⁰, Asn¹³, and Asn²⁴). The TM helices have been identified in the top view.