Sweet Taste Transduction - ACS Symposium Series (ACS Publications)

Chapter 17

Sweet Taste Transduction A Molecular-Biological Analysis

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 25, 2016 | http://pubs.acs.org Publication Date: December 31, 1991 | doi: 10.1021/bk-1991-0450.ch017

Doron Lancet and Nissim Ben-Arie Department of Membrane Research, Weizmann Institute of Science, Rehovot, Israel

While the chemistry of sweet tasting compounds has been extensively studied (1-5), precious little has been known until recently on the cellular mechanisms of sweet taste transduction. Work in the authors' laboratory, as well as in several others, has begun to shed light on this problem. Specifically, evidence has accumulated in the last three years, suggesting that sweet taste receptor proteins (as yet unidentified) activate a membrane transduction cascade. This molecular chain of events appears to be very similar to that which is associated with receptors for hormones and neurotransmitters, as well as visual photoreceptors and olfactory receptors (6-8). The proposed transduction cascade includes (see Figure 1): (1) A transmembrane protein receptor that binds sweet compounds stereospecifically and subsequently undergoes a conformational transition. (2) A membrane amplifier GTP-binding protein (G-protein) of the stimulatory type (G ). (3) The membrane enzyme adenylyl cyclase, that produces an intracellular second messenger cyclic AMP (cAMP). s

(4)

T h e e n z y m e c A M P - d e p e n d e n t protein k i n a s e (PK-A), w h i c h i s activated b y c A M P a n d catalyses protein phosphorylation. (5) A p h o s p h o r y l a t i o n - g a t e d p o t a s s i u m c h a n n e l , c l o s e d w h e n a c t e d u p o n b y P K - A , thereby leading to sensory cell depolarization a n d n e u r o t r a n s m i t t e r release. The initial elucidation of s u c h a m e c h a n i s m represents a major b r e a k t h r o u g h i n o u r u n d e r s t a n d i n g of sweet taste t r a n s d u c t i o n .

0097-6156/91/0450-0226$06.00/0 © 1991 American Chemical Society

In Sweeteners; Walters, D. Eric, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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Sweet Taste Transduction

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F i g u r e 1. A s c h e m e s h o w i n g t h e m a i n m o l e c u l a r c o m p o n e n t s o f t h e m e c h a n i s m p r o p o s e d for s w e e t t a s t e t r a n s d u c t i o n . The t a s t a n t ( s w e e t a g o n i s t ) b i n d s to a r e c e p t o r (R) a n d a c t i v a t e s i t . In its active conformation, the receptor c a n interact w i t h the G - p r o t e i n (G) a n d c a t a l y z e i t s c o n v e r s i o n t o t h e a c t i v e , G T P b o u n d form. T h e c o m p l e x of active G - p r o t e i n a n d a d e n y l y l cyclase (C) is catalytically active, generating the second messenger c A M P from ATP. c A M P b i n d s to cAMP-dependent p r o t e i n k i n a s e (PK-A) a n d s t i m u l a t e s its a c t i v i t y b y i n d u c i n g subunit dissociation. T h e active catalytic s u b u n i t of P K - A c a t a l y z e s t h e c o v a l e n t a t t a c h m e n t o f p h o s p h a t e g r o u p s (~P) t o p r o t e i n s , s p e c i f i c a l l y to a p o t a s s i u m c h a n n e l . In this case, c h a n n e l phosphorylation results i n its closure a n d a decrease i n potassium conductance (gK+K l e a d i n g to c e l l membrane depolarization. The latter m a y cause c a l c i u m influx a n d n e u r o t r a n s m i t t e r release at the s y n a p s e b e t w e e n the taste cell a n d t h e o u t g o i n g a f f e r e n t n e r v e f i b e r l e a d i n g t o t h e b r a i n . R, G a n d C are a t t a c h e d to t h e a p i c a l , m i c r o v i l l a r m e m b r a n e of t a s t e cells. P K - A is a cytoplasmic, soluble protein. The p o t a s s i u m c h a n n e l a n d the s y n a p s e are i n the b a s o l a t e r a l m e m b r a n e of the taste cell.


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CHEMORECEPTION


T h e E n i g m a o f Sweet R e c e p t i o n Sweet taste reception is u n i q u e a m o n g biological receptor mechanisms. O n one h a n d , there is obvious stereospecificity, as attested b y the fact t h a t o n l y relatively few c o m p o u n d s elicit sweet s e n s a t i o n . T h e " p h a r m a c o l o g y " of sweet taste is h i g h l y a n a l o g o u s to t h a t of d r u g s , a s w e l l a s n e u r o t r a n s m i t t e r a n d h o r m o n e a n a l o g s . S l i g h t c h e m i c a l v a r i a t i o n s l e a d to p r o n o u n c e d c h a n g e s i n s w e e t "agonist" potency, a n d often t r a n s f o r m a sweet c o m p o u n d into a bitter one. S u c h t r a n s f o r m a t i o n is r e m i n i s c e n t of t h a t f r o m a n a g o n i s t to a n a n t a g o n i s t i n c l a s s i c a l p h a r m a c o l o g y . O n the other h a n d , sweet reception probably represents the "weakest" k n o w n receptor m e c h a n i s m . The affinity toward the natural agonists, mono- a n d di-saccharides, is represented by a K d of h u n d r e d s of m i l l i m o l a r . T h i s is a p p a r e n t i n the fact t h a t the h a l f m a x i m a l e l e c t r o p h y s i o l o g i c a l , b e h a v i o r a l a n d b i o c h e m i c a l effects of s u c h s u g a r s i s a t t a i n e d a t c o n c e n t r a t i o n s b e t w e e n 0 . 1 a n d 1.0 m o l a r (9-13). N o o t h e r receptor m e c h a n i s m is k n o w n to h a v e s u c h a n attribute. Presented with such a high apparent Kd, any pharmacologist w o u l d invoke terms s u c h as "non-specific" a n d "artifactual." H o w c a n the contradiction between extreme specificity a n d very low affinity be reconciled? It i s u s e f u l t o c o n s i d e r t h e e v o l u t i o n o f s w e e t t a s t e . T h e m o s t l i k e l y e x p l a n a t i o n for the l o w a p p a r e n t affinity a s s o c i a t e d w i t h sweet taste f u n c t i o n is that the n a t u r a l ligands need be recognized at very h i g h concentration. A n o r g a n i s m satisfied w i t h very dilute s u g a r s o l u t i o n s c a n n o t fulfill its n u t r i t i o n a l needs, a n d therefore w i l l have a selective disadvantage. O n the other h a n d , chemical stereospecificity is j u s t as i m p o r t a n t here as it is i n a n y other receptor system: c o n s u m i n g the w r o n g c h e m i c a l w i l l s u r e l y be d e t r i m e n t a l . T h u s , it is possible t h a t the s p e c i a l a t t r i b u t e s of sweet taste r e c e p t i o n evolved u n d e r a u n i q u e set of evolutionary constraints, w h i c h require h i g h specificity concomitant w i t h very l o w affinity. W h a t c o u l d be the u n i q u e m o l e c u l a r p r o p e r t i e s of sweet taste receptors? A l l receptors f u n c t i o n b y w a y of m u l t i p l e " E l e m e n t a r y I n t e r a c t i o n s " (El's). The pharmacology, x-ray crystallography and m o l e c u l a r m o d e l i n g of receptor-ligand i n t e r a c t i o n s s t r o n g l y s u g g e s t that receptor specificity a n d affinity are generated through simultaneous noncovalent interactions, each bearing a small amount of b i n d i n g energy. E l ' s c o u l d be a n y of the following: hydrogen bonds, dipole-dipole interactions, ionic pair interactions, h y d r o p h o b i c i n t e r a c t i o n s a n d o t h e r w e a k i n t e r a c t i o n s , e.g., v a n d e r W a a l s f o r c e s . E a c h o f t h e s e E l ' s t y p i c a l l y c o n t r i b u t e s 1-2 k c a l / m o l e to t h e free e n e r g y c h a n g e a s s o c i a t e d w i t h l i g a n d - r e c e p t o r b i n d i n g (14). To generate a typical affinity characterized b y a d i s s o c i a t i o n c o n s t a n t K d i n t h e r a n g e o f 1 n M t o 1 μΜ, i t i s n e c e s s a r y t o h a v e 6 - 9 E l ' s . T h i s i s b a s e d o n t h e a s s u m p t i o n o f a f r e e e n e r g y o f 1.4 k c a l p e r E I , u s i n g t h e f o r m u l a for A G , t h e free e n e r g y of i n t e r a c t i o n :



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A G = - 2 . 3 - R - T - l o g K d (R i s t h e g a s c o n s t a n t ) . It i s t h e f a c t t h a t s u c h m u l t i p l e interactions are sterically ordered that confers specificity u p o n ligand-receptor binding. N o w , f o r s w e e t r e c e p t o r s , a K d o f 1 0 0 m M a m o u n t s t o o n l y one " s t a n d a r d " EIÎ T h i s , of c o u r s e , c a n n o t be r e c o n c i l e d w i t h the documented stereospecificity. Furthermore, sweet agonists (similar to other agonists) have to induce the correct c o n f o r m a t i o n a l t r a n s i t i o n i n t h e i r receptor, c o n v e r t i n g i t to t h e active state, c a p a b l e of G - p r o t e i n s t i m u l a t i o n . I n o t h e r w o r d s , i t i s n e c e s s a r y to p r o v i d e the specificity a n d a l l o s t e r i c c a p a c i t y i n t h e ligand-receptor interactions, most likely by multiple El's, without p r o d u c i n g e x c e s s i v e free e n e r g y o f b i n d i n g . O n e w a y b y w h i c h t h i s c o n t r a d i c t i o n c o u l d be reconciled is t h r o u g h the following m o d e l ( F i g u r e 2 ) : Low affinity sweet taste agonists interact with their receptors via multiple elementary interactions (El's), each having a very low free energy of binding.. S u c h a situation will obtain if the interactions are c o n s t r a i n e d so t h a t no E I is o p t i m a l . S t r a i n e d n o n covalent b o n d angles a n d distances as well as steric h i n d r a n c e effects c o u l d j o i n t l y p r o d u c e t h e d e s i r e d effect. Some El's may a c t u a l l y c o n t r i b u t e negatively to t h e b i n d i n g e n e r g y (that i s , h a v e a repulsive nature). A n o t h e r possible c o n t r i b u t i n g factor c o u l d be c o n f o r m a t i o n a l c o n s t r a i n t s , w h e r e b y t h e free e n e r g y of E l ' s i s p a r t i a l l y e x p e n d e d to i n d u c e c o n f o r m a t i o n a l t r a n s i t i o n s i n t h e receptor. A t p r e s e n t it is i m p o s s i b l e to p r e d i c t w i t h c e r t a i n t y t h e a c t u a l n u m b e r of E l ' s i n a t y p i c a l sweet receptor b i n d i n g site, b u t it is not impossible that it h a s even more E l ' s t h a n a typical d r u g receptor. T h e foregoing m o d e l h a s some interesting consequences and predictions: (1) (2)

(3)

Sweet agonists w i l l have a m u l t i p l i c i t y of f u n c t i o n a l g r o u p s . S u g a r s , w h i c h are p o l y h y d r o x y l c o m p o u n d s , are a case i n point. T h e sweet receptor b i n d i n g site w i l l be large a n d relatively complex. T h i s point is borne out b y recent developments i n t h e field o f a r t i f i c i a l s w e e t e n e r s . T h e stereospecificity r e q u i r e m e n t s for sweet c o m p o u n d s w i l l b e h a r d e r to define, a n d m o d e l s for t h e s w e e t r e c e p t o r b i n d i n g site m o r e difficult to develop. T h i s i n d e e d a p p e a r s to be the case.

G - P r o t e i n s , A d e n y l y l C y c l a s e a n d Sweet T a s t e R e c e p t i o n The first evidence that G-proteins are involved i n a n y receptor m e c h a n i s m came t h r o u g h the realization that some hormones will activate intracellular second messenger p r o d u c t i o n only i n the p r e s e n c e o f t h e n u c l e o t i d e g u a n o s i n e t r i p h o s p h a t e ( G T P ) [6,7,15). T h e m o s t w e l l k n o w n e x a m p l e of a s e c o n d m e s s e n g e r - p r o d u c i n g enzyme is a d e n y l y l cyclase, w h i c h catalyses the f o r m a t i o n of cyclic A M P (cAMP). T h i s led to the h y p o t h e s i s t h a t the receptor c o n t a i n e d a G T P b i n d i n g site, as w e l l as a n e n z y m a t i c site c a t a l y z i n g second messenger production. Later, it w a s f o u n d t h a t the



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receptor, G T P b i n d i n g entity a n d the enzyme constitute separate proteins. Subsequently, the receptor-coupled GTP binding proteins (G-proteins) were identified, purified, a n d later subjected to m o l e c u l a r c l o n i n g . G - p r o t e i n s a r e n o w k n o w n to c o n s t i t u t e a v e r y b r o a d f a m i l y ( F i g u r e 3), a n d t h e i r o c c u r e n c e s p a n s r e c e p t o r m e c h a n i s m s f r o m visual photoreceptors through brain neurotransmitters, neuropeptides, hormones, growth factors, and chemotaxis receptors. F u n c t i o n a l l y , s u c h G - p r o t e i n s are classified a c c o r d i n g to the distal m e c h a n i s m they modulate, a n d b y whether they activate or i n h i b i t s u c h m e c h a n i s m . Notable among these distal m e c h a n i s m s are the enzymes adenylate cyclase, cyclic G M P p h o s p h o d i e s t e r a s e (in v i s i o n ) , p h o s p h o l i p a s e C (the e n z y m e t h a t catalyzes p h o s p h a t i d y l i n o s i t o l turnover) a n d several types of i o n channels. M a j o r types of G - p r o t e i n s are: G , the stimulatory G-protein that activates adenylyl cyclase; G i , the inhibitory G-protein that inhibits the same enzyme; G , a brain-specific G - p r o t e i n a k i n t o G i ; t r a n s d u c i n (Gp), t h e v i s u a l G - p r o t e i n . T h e G proteins that activate phospholipase C a n d ion c h a n n e l s generally b e l o n g to the G i family. s

0

T h e m o l e c u l a r u n d e r s t a n d i n g of c h e m o s e n s o r y t r a n s d u c t i o n lagged b e h i n d t h a t of o t h e r p r o c e s s e s . A b o u t five y e a r s ago evidence b e g a n to e m e r g e t h a t a m e c h a n i s m , i n c l u d i n g a stimulatory type G-protein a n d adenylate cyclase, is central i n olfactory t r a n s d u c t i o n . T h e c u r r e n t e v i d e n c e for t h i s m e c h a n i s m i s rather extensive, a n d includes biochemical a n d m o l e c u l a r cloning r e p o r t s ( J 6 - 2 0 ) , a s w e l l a s e l e c t r o p h y s i o l o g i c a l e x p e r i m e n t s (21). M o r e r e c e n t l y , a t t e n t i o n h a s b e g u n to be d i r e c t e d t o w a r d t h e m o l e c u l a r b a s i s of taste reception. C o n s i d e r a b l e i n d i r e c t evidence h a s a c c u m u l a t e d i n the last two decades, suggesting the i n v o l v e m e n t of cyclic n u c l e o t i d e s i n sweet a n d bitter taste. This includes biochemical, histochemical and electrophysiological r e p o r t s [22-29). In the last four years experiments have b e e n c o n d u c t e d t h a t m o r e d i r e c t l y a d d r e s s the q u e s t i o n of w h e t h e r sweetener receptors could exert their action via a G-protein/adenylyl cyclase m e c h a n i s m , as detailed below. Biochemical Evidence. The first b i o c h e m i c a l experiments s u g g e s t i n g the i n v o l v e m e n t of a G - p r o t e i n i n sweet taste r e c e p t i o n [13,30) u t i l i z e d a m e m b r a n e p r e p a r a t i o n d e r i v e d f r o m r a t t o n g u e t i p s , rich i n f u n g i f o r m p a p i l l a e . N e v e r t h e l e s s , t h i s p r e p a r a t i o n i s not h i g h l y enriched i n taste cell m e m b r a n e s , since a large fraction of the t o n g u e surface is covered w i t h n o n - s e n s o r y e p i t h e l i u m , a n d the preparation contains muscle membranes as well. Furthermore, s u c h a p r e p a r a t i o n is n o t specifically e n r i c h e d i n t h a t p a r t of the t a s t e c e l l m e m b r a n e s (the a p i c a l m i c r o v i l l i ) t h o u g h t t o b e r e l a t e d t o s i g n a l t r a n s d u c t i o n . T h i s is a n o b v i o u s d r a w b a c k of t h e p r e s e n t l y available m e m b r a n e p r e p a r a t i o n s , w h e n c o m p a r e d to the m u c h purer isolated cilia preparations routinely used i n olfactory s


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Other receptors

Sweet taste receptors


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ο ο

Much weaker elementary interactions

Regular strength elementary interactions

F i g u r e 2. A h y p o t h e t i c a l m o d e l for t h e w a y b y w h i c h s w e e t receptors m a i n t a i n stereospecificity despite their very low a f f i n i t y (see t e x t ) .

G

x

Gii

Gi3

Gi2

Go

Gti

G

t

2

G

s

i

Alternative splicing products

G 2 S

G 3 S

? ? Gustatory G

s

F i g u r e 3. A tree s h o w i n g the h o m o l o g y r e l a t i o n s for different G - p r o t e i n s h e a v y (a) s u b u n i t s . T h e s t i m u l a t o r y G T P - b i n d i n g proteins are o n a separate " b r a n c h , " distinct from all others w h i c h g e n e r a l l y b e l o n g to t h e i n h i b i t o r y G T P - b i n d i n g p r o t e i n type. A t p r e s e n t , two genes are k n o w n for stimulatory G-proteins: G i w h i c h gives rise to at least four different polypeptide p r o d u c t s expressed i n different t i s s u e s , a n d G 2 (G if) w h i c h is u n i q u e l y expressed i n olfactory n e u r o n s . Two p o s s i b l e s c e n a r i o s are d e p i c t e d for the G protein involved i n s w e e t t a s t e t r a n s d u c t i o n : (1) i t i s a n e w G - p r o t e i n ["G 3"]; (2) i t is one of the k n o w n G i p r o d u c t s . s

S

0

s

S

s



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r e s e a r c h . F u t u r e d e v e l o p m e n t s w i l l h a v e to resolve t h i s p r o b l e m . A n i m p o r t a n t step forward is the d e v e l o p m e n t of a n i s o l a t e d r a t taste papillae preparation, u s e d i n conjunction w i t h a cellular c A M P a c c u m u l a t i o n a s s a y (31). T h e rat tongue tip preparation h a s a n adequate specific activity of a d e n y l y l c y c l a s e , c o m p a r a b l e to t h a t of m a n y o t h e r p r e p a r a t i o n s , e.g., l i v e r a n d e r y t h r o c y t e m e m b r a n e s . T h i s specific activity is h o w e v e r c o n s i d e r a b l y ( 1 0 0 - 1 0 0 0 fold) l o w e r t h a n t h a t s e e n i n olfactory cilia. W h e n guanine nucleotides, s u c h as G T P or its non-hydrolyzable a n a l o g G T P y S , are a d d e d , the generation of c A M P is e n h a n c e d , as expected for a s y s t e m t h a t i n c l u d e s a G - p r o t e i n . T h i s i n itself i s r a t h e r c o m m o n , a n d c a n n o t be t a k e n as evidence for receptor c o u p l i n g to a G - p r o t e i n . The crucial experiment m u s t involve a d d i t i o n of a f u n c t i o n a l l i g a n d . Here a p r o b l e m arises: the m o s t obvious sweet-tasting ligands are sugars, a n d these m u s t be u s e d at their physiological concentrations, namely 0.1-1.0 M . Planning s u c h experiments is t h u s rather tricky. The exceedingly h i g h l i g a n d c o n c e n t r a t i o n s c o u l d h a v e v a r i o u s n o n s p e c i f i c effects o n t h e m e m b r a n e e n z y m e s , effects r e l a t e d to h i g h o s m o t i c p r e s s u r e , viscosity a n d s i m p l y non-specific b i n d i n g to proteins. T h i s p r o b l e m is r e m i n i s c e n t of t h a t e n c o u n t e r e d i n the first b i o c h e m i c a l experiments w i t h odorants, where it was found that the necessary concentrations a p p r o a c h 1 m M , w h i c h c o u l d exert nonspecific e f f e c t s , e.g., o n m e m b r a n e f l u i d i t y . W h e n the experiments w i t h sugars were performed, m u c h care w a s t a k e n to c a r r y o u t t h e c o r r e c t c o n t r o l s . S e v e r a l m o n o - a n d d i s a c c h a r i d e s w e r e f o u n d to a c t i v a t e t h e t o n g u e m e m b r a n e a d e n y l y l cyclase. That s u c h activation was physiologically significant was s u p p o r t e d b y t h e f o l l o w i n g [13): (1) A c t i v a t i o n w a s o b s e r v e d o n l y i n t h e p r e s e n c e o f g u a n i n e n u c l e o t i d e s . I n t e r e s t i n g l y , b o t h G T P a n d G T P y S w e r e effective synergizers, u n l i k e m o s t other s y s t e m s , a n d s i m i l a r o n l y to turkey erythrocyte membranes. (2) S i n c e b o t h g u a n i n e n u c l e o t i d e s t e s t e d s y n e r g i z e d w i t h s u c r o s e a n d o t h e r s a c c h a r i d e s , it b e c a m e i m p o r t a n t to identify a G-protein activator that c o u l d fully activate the tongue adenylyl cyclase, irrespective of the p r e s e n c e of r e c e p t o r - a c t i v a t i n g l i g a n d s . T h i s i n d e e d w a s f o u n d to be t h e case for a l u m i n u m fluoride (AIF4-), a n o t h e r k n o w n a c t i v a t o r o f G - p r o t e i n s . This confirmed that the synergy w i t h guanine nucleotides w a s not artifactual. ( 3 ) S u g a r a c t i v a t i o n w a s t i s s u e s p e c i f i c . It w a s o b s e r v e d t o t h e f u l l extent only i n tongue tip m e m b r a n e s a n d not i n several other membrane preparations: tongue muscle, tongue non-sensory epithelium, skeletal m u s c l e a n d olfactory cilia. (4) D i f f e r e n t s u g a r s a c t i v a t e d to l a r g e l y d i f f e r e n t d e g r e e s i n t h e r a n g e of 2 0 - 1 5 0 % . T h i s is i n c o n s i s t e n t w i t h a n y e x p l a n a t i o n i n t e r m s of n o n s p e c i f i c , p h y s i c a l effects.


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T h e o r d e r of p o t e n c y of t h r e e different s u g a r s ( m a l t o s e < g l u c o s e < s u c r o s e ) w a s s i m i l a r to t h a t p r e v i o u s l y o b t a i n e d b y electrophysiological recordings from the rat c h o r d a t y m p a n i n e r v e (10-12). (6) C e r t a i n b i v a l e n t c a t i o n s ( C u a n d Z n ) i n h i b i t the activation of a d e n y l y l c y c l a s e b y s u c r o s e , i n p a r a l l e l t o t h e i r i n h i b i t o r y effect i n e l e c t r o p h y s i o l o g i c a l r e c o r d i n g s (32,33). (7) T h e s u g a r a n a l o g methyl-4,6-dichloro-4,6-dideoxygalactop y r a n o s i d e i n h i b i t s the a c t i v a t i o n of a d e n y l y l c y c l a s e b y s u c r o s e , a g a i n i n p a r a l l e l to its i n h i b i t i o n of electrop h y s i o l o g i c a l l y r e c o r d e d r e s p o n s e s (34). A s t r o n g c o r r o b o r a t i o n for the s i g n i f i c a n c e of t h e c A M P m e c h a n i s m i n sweet taste comes from the more recent results obtained i n isolated rat papillae, monitoring c A M P generation i n intact cells. H i g h l y s i g n i f i c a n t e n h a n c e m e n t of s e c o n d m e s s e n g e r generation w a s o b s e r v e d i n r e s p o n s e t o e x t r a c e l l u l a r s u c r o s e (31).


+ 2

+ 2

T h e Possible Nature of the G u s t a t o r y G-Protein and A d e n y l y l C y c l a s e . T h e fact t h a t s u g a r s activate a d e n y l y l cyclase r a t h e r t h a n i n h i b i t i t s u g g e s t s t h e p a r t i c i p a t i o n of a G - p r o t e i n b e l o n g i n g to t h e s t i m u l a t o r y ( G ) c l a s s . It i s p r e s e n t l y u n k n o w n w h i c h s u b t y p e o f G is c o u p l e d to sweet taste receptors. Olfactory reception utilizes a novel, specific G-protein, k n o w n as G 2 or G if. T h i s protein is c o d e d for b y a s e p a r a t e gene, h i g h l y (-90%) h o m o l o g o u s to, b u t different f r o m , the G i gene. G i c o d e s for s e v e r a l p o l y p e p t i d e products generated by a process k n o w n as alternative mRNA s p l i c i n g , a n d expressed i n m a n y different t i s s u e s . T h e b i o c h e m i c a l properties of the putative gustatory G are different f r o m those of G 2 (Golf): t h e l a t t e r i s s t r o n g l y a c t i v a t e d b y b o t h G T P y S a n d A 1 F (35,36), w h i l e t h e f o r m e r s h o w s p a r t i a l a c t i v a t i o n w i t h a g o n i s t s y n e r g y for G T P y S a n d full a c t i v a t i o n o n l y w i t h A 1 F - (13). It i s p o s s i b l e t h a t g u s t a t o r y G i s c o d e d for b y a n e w gene, b u t m o r e l i k e l y i t i s d e r i v e d f r o m t h e G i g e n e ( F i g u r e 3). If t h e l a t t e r p o s s i b i l i t y h o l d s , g u s t a t o r y G m a y be i d e n t i c a l to o n e of t h e g e n e p r o d u c t s expressed i n other tissues, or a n e w protein variant. M o l e c u l a r c l o n i n g w i l l allow r e s o l u t i o n of t h i s p r o b l e m . s

s

S

s

0

s

s

4

S

4

s

s

s

A n intriguing correlation w i t h the notion that a t r a n s d u c t i o n m e c h a n i s m a k i n to t h a t for h o r m o n e r e c e p t o r s i s i n v o l v e d i n s w e e t taste r e s p o n s e s i s afforded b y a report s h o w i n g t h a t a n i n a b i l i t y to taste sweet (aglycogeusia) is associated w i t h a metabolic genetic d i s e a s e , p s e u d o h y p o p a r a t h y r o i d i s m (37). Future investigations s h o u l d d e t e r m i n e t h e e x a c t m o l e c u l a r b a s i s for t h i s r a r e deficiency. Incidentally, the report that pseudohypoparathyroidism type l a , a r i s i n g f r o m a G i d e f i c i e n c y (38), is a s s o c i a t e d w i t h o l f a c t o r y deficits, c a n n o t s i m p l y be explained b y a b r e a k i n the p e r i p h e r a l chemosensory transduction cascade. T h i s is i n v i e w of the e v i d e n c e t h a t o l f a c t o r y G - p r o t e i n i s c o d e d b y a d i f f e r e n t g e n e , G 2s

S


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CHEMORECEPTION

T h u s , a n e x p l a n a t i o n i n t e r m s of the c e n t r a l n e r v o u s s y s t e m s h o u l d be favored. No i n f o r m a t i o n is available o n the n a t u r e of g u s t a t o r y a d e n y l y l cyclase. The enzyme h a s recently been identified i n several tissues b y a c o m b i n a t i o n of a n t i b o d y b i n d i n g , p u r i f i c a t i o n a n d m o l e c u l a r cloning. B r a i n h a s two major types: a 110 k D a l t o n calmodulinsensitive form a n d a 150 k D a l t o n calmodulin-insensitive form. Olfactory cilia have their u n i q u e form, AC if, w h i c h is 180 k D a l t o n i n s i z e (39). T h e t a s t e s y s t e m m a y h a v e o n e o f t h e k n o w n f o r m s o r a novel one.


0

G u s t a t o r y Ion C h a n n e l M o d u l a t i o n b y c A M P A s s u m i n g that c A M P is a second messenger for sweet taste responses, one m a y w o n d e r h o w changes i n this cytoplasmic compound lead to t h e known taste cell responses, i.e., d e p o l a r i z a t i o n a n d r e l e a s e of n e u r o t r a n s m i t t e r . At present, answers to t h i s q u e s t i o n r e l y s o l e l y o n r e c e n t single cell e l e c t r o p h y s i o l o g i c a l r e c o r d i n g s (40-42). c A M P w a s f o u n d t o i n d u c e t r a n s m e m b r a n e d e p o l a r i z a t i o n s i m i l a r to t h a t c a u s e d b y sweet tastants s u c h as sucrose. T h i s d e p o l a r i z a t i o n w a s f o u n d to be i n d u c e d b y the c l o s u r e of 4 4 p i c o S i e m e n p o t a s s i u m c h a n n e l s i n the a p i c a l m e m b r a n e s of taste cells. T h e m o l e c u l a r b a s i s for s u c h c A M P c h a n n e l conductance m o d u l a t i o n h a s also been established b y p a t c h clamp recordings. T h i s effect i s b l o c k e d b y t h e W a l s h - K r e b s p r o t e i n k i n a s e i n h i b i t o r , k n o w n to i n a c t i v a t e cAMP-dependent p r o t e i n k i n a s e . F u r t h e r m o r e , the active catalytic s u b u n i t of s u c h k i n a s e c a n m i m i c t h e effect o f c A M P o r t a s t a n t . T h i s l e n d s s t r o n g s u p p o r t to the l a s t steps i n the t r a n s d u c t i o n m e c h a n i s m s h o w n i n F i g u r e 1. T h e m e c h a n i s m i s c l e a r l y d i f f e r e n t f r o m t h a t f o u n d i n olfactory s e n s o r y n e u r o n s , w h e r e c A M P d i r e c t l y b i n d s to c a t i o n c h a n n e l s a n d i n d u c e s t h e i r o p e n i n g (43). S u m m a r y and Prospects O u r u n d e r s t a n d i n g of the m o l e c u l a r b a s i s of s w e e t taste r e c e p t i o n h a s p r o c e e d e d t r e m e n d o u s l y i n t h e l a s t few y e a r s . A d d e d to a b u r g e o n i n g d i s c i p l i n e r e l a t e d to the fine s t r u c t u r e of s w e e t e n e r s a n d t h e i r b i n d i n g site(s) i s a v i e w of t h e e n z y m a t i c cascade activated b y gustatory receptor proteins i n sweet-sensitive taste cells. T h e s e two b o d i e s of k n o w l e d g e s h o u l d c o m p l e m e n t each other, a n d l e a d to a n e m e r g i n g field of "taste p h a r m a c o l o g y , " a k i n to t h a t w h i c h h a s l o n g e x i s t e d for the a c t i o n of d r u g s a n d hormones. A most awaited development is the future identification, i s o l a t i o n a n d c h a r a c t e r i z a t i o n of the p r o t e i n receptors themselves. It i s m o s t l i k e l y t h a t t h e r e c e p t o r s f o r s w e e t c o m p o u n d s w i l l t u r n o u t to b e h o m o l o g o u s to o t h e r r e c e p t o r s k n o w n to b e c o u p l e d to G - p r o t e i n s (cf. réf. 18). E x a m p l e s are r h o d o p s i n , the v i s u a l



17. LANCET & BEN-ARIE


235

p h o t o r e c e p t o r , a - a n d β-adrenergic r e c e p t o r s , m u s c a r i n i c a c e t y l choline receptors, dopamine receptors a n d many other neurotransmitter a n d neuropeptide receptors. A l l of these share a structure that includes seven transmembrane helical segments, extracellular glycosylation a n d numerous intracellular p h o s p h o r y l a t i o n sites (44). Sweet taste receptors m a y belong to the same receptor superfamily. Some of the molecular-biological strategies that are currently u s e d for attempting t h e isolation of genes coding for sweet taste s y s t e m receptors a r e b a s e d o n t h i s n o t i o n . W h e n isolated, s u c h genes c o u l d reveal the fine s t r u c t u r e o f the sweet c o m p o u n d b i n d i n g site, a s well a s t h e possible multiplicity of sweet taste receptors, a n d a i d i n future development of n e w n o n - n u t r i t i v e sweeteners.

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26. Nomura, H.; Asanuma, N. Chem. Senses 1982, 7, 71-80. 27. Nagahama, S.; Kobatake, Y.; Kurihara, K. J. Gen.Physiol.1982, 80, 785-800. 28. Esakov, A.I.; Mescherjakova, O.D. Chem. Senses 1984, 8, 329339. 29. Wieczorek, H.; Schweikl, H. Insect Biochem. 1985, 15, 723728. 30. Lancet, D.; Striem, B.J.; Pace, U.; Zehavi, U.; Naim, M. Soc. Neurosci. Abstr. 1987, 31, 361. 31. Striem, B.J.; Naim, M.; Lindemann, B. Abstracts of Papers, European Chemoreception Research Organization IX Congress, Netherlands, 1990;p97. 32. Yamamoto, T.; Kawamura, Y. J. Osaka Univ. Dental Sch. 1971, 11, 99-104. 33. Kasahara, T.; Iwasaki, K.; Sato, M. Chem. Senses 1987, 12, 295-305. 34. Striem, B.J.; Yamamoto, T.; Naim, M.; Lancet, D.; Jakinovich, W.J.; Zehavi, U. Chem. Senses, in press. 35. Pace, U.; Hanski, E.; Salomon, Y.; Lancet, D. Nature 1985, 316, 255-258. 36. Pace, U.; Lancet, D. Proc.Natl.Acad. Sci. USA 1986, 83, 4947-4951. 37. Henkin, R.I.; Shallenberger, R.S. Nature 1970, 227, 965-966. 38. Weinstock, R.S.; Wright, H.N.; Spiegel, A.M.; Levine, M.A.; Moses, A.M. Nature 1986, 322, 635-636. 39. Pfeuffer, E.; Mollner, S.; Lancet, D.; Pfeuffer, T. J. Biol. Chem. 1989, 264, 18803-18807. 40. Avenet, P.; Hofmann, F.; Lindemann, B. Nature 1988, 331, 351-354. 41. Avenet, P.; Hofmann, F.; Lindemann, B. Comp. Biochem. Physiol. [a] 1988, 90, 681-685. 42. Tonosaki, K.; Funakoshi, M. Nature 1988, 331, 354-356. 43. Nakamura, T.; Gold, G.H. Nature 1987, 325, 442-444. 44. Dohlman, H.G.; Caron, M.G.; Lefkowitz, R.J. Biochemistry 1987, 26, 2657-2664. RECEIVED September 28, 1990


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