Signal Transduction
In comparison with other major protein families, the level of sequence homology between the members of the huge superfamily of 7TM receptors is low. However, sequence alignments do reveal some shared traits that form the basis for a classification into families.
Categories of 7TM receptor
A large proportion of the proteins acknowledged as members of the 7TM receptor family are orphans, having no known ligand and hence no known function. In addition, a similarly large proportion of the genes that code for 7TM proteins are not expressed. If we include those coding for 7TM proteins present in olfactory epithelial cells (probable receptors for odorant
substances), then this family is truly enormous, approaching 2000 members, or 5% of the mammalian genome.
While the general structural plan of these proteins seems assured, there are of course important differences. The huge variety of activating ligands, encompassing ions (Ca2 ), small molecules, and proteins, makes this selfevident (Table 3.3). One extreme example is that of rhodopsin, which binds its ligand, 11-cis retinal, covalently (see Chapter 6). Unlike rhodopsin, the
receptors for hormones exist in both free and bound states. The rate at which they can be activated must be limited by the availability of the ligand, its
Table 3.3 Categories of 7TM receptors
Receptor properties |
Ligands |
|
|
Ligand binds in the core region of the 7 |
11-cis-retinal (in rhodopsin) |
transmembrane helices |
acetylcholine |
|
catecholamines |
|
biogenic amines (histamine, serotonin, etc.) |
|
nucleosides and nucleotides |
|
leukotrienes, prostaglandins, prostacyclins, thromoboxanes |
Short peptide ligands bind partially in the core region and to the external loops
peptide hormones (ACTH, glucagon, growth hormone) parathyroid hormone, calcitonin
Ligands make several contacts with the |
hypothalamic glycoprotein releasing factors (TRH, GnRH) |
N-terminal segment and the external loops |
|
|
|
Induce an extensive reorganization of an |
metabotropic receptors for neurotransmitters (such as GABA and |
extended N-terminal segment |
glutamate) |
|
Ca2 -sensing receptors, for example on parathyroid cells, thyroidal |
|
C-cells (which secrete calcitonin) and on the renal juxtaglomerular |
|
apparatus |
|
|
Proteinase activated receptors |
receptors for thrombin and trypsin |
|
|
58
Receptors
rate of diffusion into the binding site and then its rate of attachment. What happens next is now well recognized. The activated receptors interact with G proteins and thence with intracellular effector enzymes to generate or mobilize second messengers (such as cAMP, Ca2 , etc.). However, at the time that cAMP and its synthesizing enzyme adenylyl cyclase were discovered, understanding of the receptors was purely conceptual. No receptor proteins had been isolated and knowledge of membrane structure was very limited. Whether or not receptors and the enzyme activities that they control comprise a single entity had not been resolved.39
Receptor diversity: variation and specialization
Most signalling proteins with homologues in different species (orthologues) exhibit sequence homology, for example GTP-binding proteins, PKA, and phospholipase C in mammals, invertebrates, and yeast. The 7TM receptors, by contrast, have few conserved residues. Even within the recognized subfamilies (such as the rhodopsin-like receptors discussed below), the extent of sequence homology is exceedingly limited. Indeed, the idea that they are all derived from a single common ancestor or whether they arose as the result of convergent evolutionary processes has been repeatedly discussed. Moreover, if divergence in the face of conserved architecture is evident for the membrane-spanning regions, it is even more obvious for the various ligand-binding domains.
For example, within just one family, the adhesion receptors, the exposed segments vary in length from 7 up to about 2800 residues.41
Although it is possible to classify these receptors on the basis of the types of ligands which they bind (see Figure 3.17), this bears little relationship to their evolutionary ancestry. Phylogenetic analysis can be used to investigate this, but it relies on single amino acid changes in conserved regions and
it is unwise to base calculations on sections that are highly variable and may have undergone domain addition or replacement (see Chapter 24). In consequence, analyses of 7TM proteins have generally been based on the
common, conserved transmembrane segments. Such an approach, applied to all the putative 7TM receptor proteins recognized in the year 1994, proposed six ‘clans’,42,43 some of which do not exist in animals. Later, with the advent of the human genome database, a more searching analysis of the 7TM receptorlike proteins became possible. It has been concluded that there are four main human classes: A, B, C, and Frizzled (Table 3.4). The phylogenetic relationships of receptors that form these classes, based on their transmembrane segments, are illustrated in Figure 3.14. This includes a fifth class, the adhesion family, emerging from a group of mostly orphan receptors within Class B.44
The rhodopsin class is the largest and is illustrated separately in Figure 3.15.
From these analyses, it seems certain that the members within each family do share a common evolutionary origin. It is clear, too, that the total number of
As late as 1973, Raymond Ahlquist, who made the distinction between
- and -adrenergic receptors, still expressed his doubts about their actual existence with the words:
This would be true if I were so presumptuous as to believe that and receptors really did exist. There are those that think so and even propose to describe their intimate structure. To me they
are an abstract concept conceived to explain observed responses of tissues produced by chemicals of various structures.40
59
Signal Transduction
Table 3.4 Classification of 7TM receptors
Main classes |
Number |
Comments |
Ligands |
|
|
|
|
Class A |
271 388 |
There are four main non-olfactory subgroups |
prostaglandins |
Rhodopsin |
olfactory |
Nearly all share common motifs such as DRY |
thromboxanes |
family |
|
(D/E-R-Y/F) between transmembrane segment |
serotonin |
|
|
II and inner loop 2 |
histamine (H2) |
|
|
Ligand binding occurs mostly in the cavity |
catecholamines |
|
|
between the transmembrane segments |
acetylcholine |
|
|
(glycoprotein receptors are exceptions) |
rhodopsin |
|
|
|
cone opsins |
|
|
|
melanopsin |
|
|
|
melatonin |
|
|
|
|
Class B |
15 |
Generally bind large polypeptides that share |
glucagons |
Secretin family |
|
considerable sequence identity. Often acting |
GnRH |
|
|
in a paracrine manner. N-terminal sequences |
PTH |
|
|
(60–80 residues) contain conserved sulfhydryl |
CRF |
|
|
bridges |
secretin |
|
|
|
|
Class C |
22 |
The N-terminus forms two distinct lobes. Said |
glutamate, GABA Ca2 |
Glutamate and |
|
to resemble a ‘Venus fly trap’ which snaps |
sweet tastes |
GABA family |
|
around the bound ligand |
(TAS1 subgroup) |
|
|
|
|
Frizzled class |
11 frizzled |
Includes the mammalian counterparts of |
Wnt |
|
25 TAS2 |
the frizzled receptor of Drosophila and Wnt |
Hedgehog |
|
|
receptors |
TAS2 stimuli |
|
|
Also taste receptors of the TAS2 subgroup, |
|
|
|
possibly sensitive to bitter tastes |
|
|
|
|
|
Adhesion |
33 |
Only recently recognized as a family distinct |
mostly unidentified |
family |
|
from the secretin group. |
CD55 (regulates |
|
|
The N-terminal 200–2800 residues are often |
complement assembly) |
|
|
rich in glycosylation sites, proline residues, |
chondroitin sulfate |
|
|
mucin stalks, and motifs expected to take |
|
|
|
part in cell adhesion. Includes brain-specific |
|
|
|
angiogenesis inhibitor, CELSR (cadherin- |
|
|
|
EGF LAG receptor), and EMR (EGF-like module- |
|
|
|
containing mucin-like hormone receptor) |
|
|
|
present on macrophages and other cells of the |
|
|
|
immune system |
|
|
|
|
|
7TM receptors is very large. A recent study of known 7TM proteins, avoiding polymorphisms and redundancies has identified a dataset of 791 unique, full length human receptors, of which some 400 are non-olfactory.45 Extensive information is published at http://www.iuphar-db.org/index.jsp and http:// www.gpcr.org/7tm/. An olfactory receptor database is provided at http:// senselab.med.yale.edu/ordb/default.asp.
60
Receptors
Fig 3.14 Phylogenetic relationship between the 7TM receptor-like proteins in the human genome: secretin, glutamate, Frizzled, and adhesion families. EMR, egf module-containing receptors; CELSR, cadherin-EGF LAG receptor.
Adapted from Fredriksson et al.44
Binding of low-molecular-mass ligands
The binding sites for most low-molecular-mass ligands such as acetylcholine, those derived from amino acids (catecholamines, histamine, etc.) and the eicosanoids (see Table 2.1, page 22) are located deep within the hydrophobic cores of their receptors (see Figure 3.17a). Attachment of catecholamines is understood to involve hydrogen bonding of the two catechol OH groups to serine residues 204 and 207 on the fifth membrane spanning (E) -helix (Figure 3.16, also Figure 3.13) and by electrostatic interaction of the amine group
to an aspartate residue (113) on the C helix. The binding of acetylcholine similarly forms a cross-link between two membrane-spanning helices of muscarinic receptors. In this way, the bound hormone molecules are oriented
61
Signal Transduction
Fig 3.15 Phylogenetic relationship between the rhodopsin-like 7TM receptor proteins in the human genome.
Adapted from Fredriksson et al.44
in the plane parallel to the membrane. They form bridges between two transmembrane spans of their receptors, perturbing their orientation relative to each other. This is the origin of the signal that is conveyed to the cell.-Blockers, such as propranolol, also bind at this location with high affinity and so impede the access of activating ligands. However, lacking the catechol group, they fail to establish the critical link between membrane spanning segments. They fail to activate the receptor.
In contrast to the receptors for low-molecular-mass ligands, the binding sites for peptide hormones such as ACTH and glucagon are situated on the
62